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At least 451 records · Page 25
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels

Here, the filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Differential Gene Expression in A Cross-Feeding Two-Species Model Microbial Community Under Simulated Microgravity and Deep-Space Radiation

A long-term goal of space biology is to understand interspecies microbial interactions in space. Presently, little is known about the combined effect of microgravity and ionizing radiation on bacterial community response when species are interdependent through exchange of metabolites in fluid medium (cross-feeding). Microgravity is expected to slow interspecies mass transfer and growth in cross-feeding communities in the low-shear, diffusion-limited environment, while ionizing radiation may influence stress response to direct (DNA damage) and indirect damage (ROS). Using a well-understood, two-species (Escherichia coli and Salmonella enterica) microbial community engineered to be a model for studying cross-feeding, we simulated galactic cosmic rays (GCRsim) and microgravity to test the hypothesis: exposure to ionizing radiation causes cell damage or stress, altering transcriptomic community responses in metabolically interdependent cells, which is exacerbated by microgravity. We expect to see differential gene expression between cross-feeding and non-cross-feeding communities. We measured GCRsim effects on growth and gene expression in well-mixed versus simulated-microgravity conditions and in cross-feeding and non-cross-feeding medium. Microbial cultures were inoculated into liquid medium in rotating wall vessels (RWV) with different rotation rates: 5 RPM (simulated microgravity) and 50 RPM (well-mixed). The E. coli-S. enterica consortium, under simulated microgravity, were exposed to 500 mGy of Simplified 5-ion Galactic Cosmic Ray Simulation for 2 hours at Brookhaven National Lab. We harvested samples 40 minutes after irradiation for extraction and sequencing (NASA GeneLab). Here we present the differential gene expression analysis results, which reveal altered transcriptomic community responses, even where growth rate differences are not observed. Gene expression of these actively metabolizing microbial communities in GCRsim may illuminate molecular mechanisms of microbial interactions in space. Understanding how microbial community gene expression, metabolism, and other cellular processes are influenced by spaceflight stressors can inform the use of microbes in human life support for low Earth orbit missions and beyond.

microgravity↗
DOE OSTI2021

Epigenetic Control of Drought Response in Sorghum (EPICON)

Genetic manipulation of crops to increase the presence of desirable traits has been critical to increasing agricultural productivity. These changes have primarily involved modification of the plant’s DNA sequence. However, there is increasing evidence that environmental responses are also mediated by epigenetics, which involves heritable changes without changes in DNA sequence. Epigenetic changes have been shown to play a major role in regulating plant responses to drought, an increasing problem worldwide due to climate change. In general, exposure of plants to water limitation triggers epigenetic changes, which include remodeling of chromatin, the network of DNA, RNA and various proteins making up chromosomes, and related changes in regulatory mechanisms. EPICON’s efforts focus on unraveling the role epigenetic signals play in acclimation to and recovery from drought through effects on individual transcription factors or transcriptional networks that direct entire metabolic pathways. To achieve this goal we will follow responses to water deprivation in sorghum, a widely cultivated cereal with recognized drought tolerance. In EPICON’s field trials, sorghum will be grown under controlled irrigation conditions. Leaf and root samples will be taken to perform molecular phenotyping to track changes in epigenetic, transcriptomic, metabolomic and proteomic footprints. Analysis of this data will provide a better understanding of the epigenetic processes related to drought tolerance, leading to our ultimate goal of identifying transcriptional regulators and pathways controlling drought resistance. The identified genetic targets and their regulatory pathways will be used in future efforts to improve growth of sorghum and other crops in the field and in marginal lands under water-limiting conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

Here, quantitative gene expression analysis in intact single cells can be achieved using single molecule-based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2 and their ratio ( Nkx2 - 2/Ins2 ). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct on-time fractions, laying the foundation for reliable single-cell transcriptomics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Comparative Transcriptomics Among Four White Pine Species

Conifers are the dominant plant species throughout the high latitude boreal forests as well as some lower latitude temperate forests of North America, Europe, and Asia. As such, they play an integral economic and ecological role across much of the world. This study focused on the characterization of needle transcriptomes from four ecologically important and understudied North American white pines within the Pinus subgenus Strobus. The populations of many Strobus species are challenged by native and introduced pathogens, native insects, and abiotic factors. RNA from the needles of western white pine (Pinus monticola), limber pine (Pinus flexilis), whitebark pine (Pinus albicaulis), and sugar pine (Pinus lambertiana) was sampled, Illumina short read sequenced, and de novo assembled. The assembled transcripts and their subsequent structural and functional annotations were processed through custom pipelines to contend with the challenges of non-model organism transcriptome validation. Orthologous gene family analysis of over 58,000 translated transcripts, implemented through Tribe-MCL, estimated the shared and unique gene space among the four species. This revealed 2025 conserved gene families, of which 408 were aligned to estimate levels of divergence and reveal patterns of selection. Specific candidate genes previously associated with drought tolerance and white pine blister rust resistance in conifers were investigated.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa . Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Silencing of OSBP-related protein 8 (ORP8) modifies the macrophage transcriptome, nucleoporin p62 distribution, and migration capacity

ORP8 is an oxysterol/cholesterol binding protein anchored to the endoplasmic reticulum and the nuclear envelope, and is abundantly expressed in the macrophage. We created and characterized mouse RAW264.7 macrophages with ORP8 stably silenced using shRNA lentiviruses. A microarray transcriptome and gene ontology pathway analysis revealed significant alterations in several nuclear pathways and ones associated with centrosome and microtubule organization. ORP8 knockdown resulted in increased expression and altered subcellular distribution of an interaction partner of ORP8, nucleoporin NUP62, with an intranuclear localization aspect and association with cytoplasmic vesicular structures and lamellipodial edges of the cells. Moreover, ORP8 silenced cells displayed enhanced migration, and a more pronounced microtubule cytoskeleton than controls expressing a non-targeting shRNA. ORP8 was shown to compete with Exo70 for interaction with NUP62, and NUP62 knockdown abolished the migration enhancement of ORP8-silenced cells, suggesting that the endogenous ORP8 suppresses migration via binding to NUP62. As a conclusion, the present study reveals new, unexpected aspects of ORP8 function in macrophages not directly involving lipid metabolism, but rather associated with nuclear functions, microtubule organization, and migration capacity. -- Highlights: Black-Right-Pointing-Pointer The phenotype of Raw264.7 macrophage with ORP8 silenced is characterized. Black-Right-Pointing-Pointer ORP8 silencing alters mRNA levels of nuclear and microtubule/centrosome pathways. Black-Right-Pointing-Pointer ORP8 silencing results in increased expression and altered distribution of NUP62. Black-Right-Pointing-Pointer ORP8 silenced macrophages show enhanced migration and altered microtubule cytoskeleton. Black-Right-Pointing-Pointer ORP8 competes in vitro with Exo70 for binding to NUP62.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Deciphering triterpenoid saponin biosynthesis by leveraging transcriptome response to methyl jasmonate elicitation in Saponaria vaccaria

Abstract Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria . This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP- d -fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis

Summary Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure–function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4‐β‐xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4‐β‐xylan formation, XYS 1/ IRX 10 in plants, IRX 7, IRX 8, IRX 9, IRX 14 and GUX occurred for the first time in charophyte algae. An XYS 1/ IRX 10 ortholog from Klebsormidium flaccidum , designated K. flaccidum XYLAN SYNTHASE ‐1 ( Kf XYS 1), possesses 1,4‐β‐xylan synthase activity, and 1,4‐β‐xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4‐β‐xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide‐based plant cell walls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A shift between mineral and nonmineral sources of iron and sulfur causes proteome-wide changes in Methanosarcina barkeri

ABSTRACT Iron (Fe) and sulfur (S) are required elements for life, and changes in their availability can limit the ecological distribution and function of microorganisms. In anoxic environments, soluble Fe typically exists as ferrous iron [Fe(II)] and S as sulfide (HS − ). These species exhibit a strong affinity that ultimately drives the formation of sedimentary pyrite (FeS 2 ). Recently, paradigm-shifting studies indicate that Fe and S in FeS 2 can be made bioavailable by methanogens through a reductive dissolution process. However, the impact of the utilization of FeS 2 , as opposed to canonical Fe and S sources, on the phenotype of cells is not fully understood. Here, shotgun proteomics was utilized to measure changes in the phenotype of Methanosarcina barkeri MS grown with FeS 2 , Fe(II)/HS − , or Fe(II)/cysteine. Shotgun proteomics tracked 1,019 proteins overall, with 307 observed to change between growth conditions. Functional characterization and pathway analyses revealed these changes to be systemic and largely tangential to Fe/S metabolism. As a final step, the proteomics data were viewed with respect to previously collected transcriptomics data to deepen the analysis. Presented here is evidence that M. barkeri adopts distinct phenotypes to exploit specific sources of Fe and S in its environment. This is supported by observed protein abundance changes across broad categories of cellular biology. DNA adjacent metabolism, central carbon metabolism methanogenesis, metal trafficking, quorum sensing, and porphyrin biosynthesis pathways are all features in the phenotypic differentiation. Differences in trace metal availability attributed to complexation with HS − , either as a component of the growth medium [Fe(II)/HS − ] or generated through reduction of FeS 2 , were likely a major factor underpinning these phenotypic differences. IMPORTANCE The methanogenic archaeon Methanosarcina barkeri holds great potential for industrial bio-mining and energy generation technologies. Much of the biochemistry of this microbe is poorly understood, and its characterization will provide a glimpse into biological processes that evolved close to life’s origin. The discovery of its ability to extract iron and sulfur from bulk, solid-phase minerals shifted a longstanding paradigm that these elements were inaccessible to biological systems. The full elucidation of this process has the potential to help scientists and engineers extract valuable metals from low-grade ore and mine waste generating energy in the form of methane while doing so.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

The WRKY transcription factor family and senescence in switchgrass

Early aerial senescence in switchgrass (Panicum virgatum) can significantly limit biomass yields. WRKY transcription factors that can regulate senescence could be used to reprogram senescence and enhance biomass yields. All potential WRKY genes present in the version 1.0 of the switchgrass genome were identified and curated using manual and bioinformatic methods. Expression profiles of WRKY genes in switchgrass flag leaf RNA-Seq datasets were analyzed using clustering and network analyses tools to identify both WRKY and WRKY-associated gene co-expression networks during leaf development and senescence onset. We identified 240 switchgrass WRKY genes including members of the RW5 and RW6 families of resistance proteins. Weighted gene co-expression network analysis of the flag leaf transcriptomes across development readily separated clusters of co-expressed genes into thirteen modules. A visualization highlighted separation of modules associated with the early and senescence-onset phases of flag leaf growth. The senescence-associated module contained 3000 genes including 23 WRKYs. Putative promoter regions of senescence-associated WRKY genes contained several cis-element-like sequences suggestive of responsiveness to both senescence and stress signaling pathways. A phylogenetic comparison of senescence-associated WRKY genes from switchgrass flag leaf with senescence-associated WRKY genes from other plants revealed notable hotspots in Group I, IIb, and IIe of the phylogenetic tree. We have identified and named 240 WRKY genes in the switchgrass genome. Twenty three of these genes show elevated mRNA levels during the onset of flag leaf senescence. Eleven of the WRKY genes were found in hotspots of related senescence-associated genes from multiple species and thus represent promising targets for future switchgrass genetic improvement. Overall, individual WRKY gene expression profiles could be readily linked to developmental stages of flag leaves.

Biotechnology & Applied Microbiology↗
DOE OSTI2018

Gene Gangs of the Chloroviruses: Conserved Clusters of Collinear Monocistronic Genes

Chloroviruses (family Phycodnaviridae) are dsDNA viruses found throughout the world’s inland waters. The open reading frames in the genomes of 41 sequenced chloroviruses (330 ± 40 kbp each) representing three virus types were analyzed for evidence of evolutionarily conserved local genomic “contexts”, the organization of biological information into units of a scale larger than a gene. Despite a general loss of synteny between virus types, we informatically detected a highly conserved genomic context defined by groups of three or more genes that we have termed “gene gangs”. Unlike previously described local genomic contexts, the definition of gene gangs requires only that member genes be consistently co-localized and are not constrained by strand, regulatory sites, or intervening sequences (and therefore represent a new type of conserved structural genomic element). An analysis of functional annotations and transcriptomic data suggests that some of the gene gangs may organize genes involved in specific biochemical processes, but that this organization does not involve their coordinated expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Diversification of multipotential postmitotic mouse retinal ganglion cell precursors into discrete types

The genesis of broad neuronal classes from multipotential neural progenitor cells has been extensively studied, but less is known about the diversification of a single neuronal class into multiple types. We used single-cell RNA-seq to study how newly born (postmitotic) mouse retinal ganglion cell (RGC) precursors diversify into ~45 discrete types. Computational analysis provides evidence that RGC transcriptomic type identity is not specified at mitotic exit, but acquired by gradual, asynchronous restriction of postmitotic multipotential precursors. Some types are not identifiable until a week after they are generated. Immature RGCs may be specified to project ipsilaterally or contralaterally to the rest of the brain before their type identity emerges. Optimal transport inference identifies groups of RGC precursors with largely nonoverlapping fates, distinguished by selectively expressed transcription factors that could act as fate determinants. Our study provides a framework for investigating the molecular diversification of discrete types within a neuronal class.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Mechanism of H 2 S Oxidation by the Dissimilatory Perchlorate-Reducing Microorganism Azospira suillum PS

The genetic and biochemical basis of perchlorate-dependent H 2 S oxidation (PSOX) was investigated in the dissimilatory perchlorate-reducing microorganism (DPRM) Azospira suillum PS (PS). Previously, it was shown that all known DPRMs innately oxidize H 2 S, producing elemental sulfur (S o ). Although the process involving PSOX is thermodynamically favorable (ΔG ° ' = -206 kJ ∙ mol -1 H 2 S), the underlying biochemical and genetic mechanisms are currently unknown. Interestingly, H 2 S is preferentially utilized over physiological electron donors such as lactate or acetate although no growth benefit is obtained from the metabolism. Here, we determined that PSOX is due to a combination of enzymatic and abiotic interactions involving reactive intermediates of perchlorate respiration. Using various approaches, including barcode analysis by sequencing (Bar-seq), transcriptome sequencing (RNA-seq), and proteomics, along with targeted mutagenesis and biochemical characterization, we identified all facets of PSOX in PS. In support of our proposed model, deletion of identified upregulated PS genes traditionally known to be involved in sulfur redox cycling (e.g., Sox, sulfide:quinone reductase [SQR]) showed no defect in PSOX activity. Proteomic analysis revealed differential abundances of a variety of stress response metal efflux pumps and divalent heavy-metal transporter proteins, suggesting a general toxicity response. Furthermore, in vitro biochemical studies demonstrated direct PSOX mediated by purified perchlorate reductase (PcrAB) in the absence of other electron transfer proteins. The results of these studies support a model in which H 2 S oxidation is mediated by electron transport chain short-circuiting in the periplasmic space where the PcrAB directly oxidizes H 2 S to S o . The biogenically formed reactive intermediates (ClO 2 - and O 2 ) subsequently react with additional H 2 S, producing polysulfide and S o as end products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

A comprehensive collection of systems biology data characterizing the host response to viral infection

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). As a result, by comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Data integration reveals key homeostatic mechanisms following low dose radiation exposure

The goal of this study was to define pathways regulated by low dose radiation to understand how biological systems respond to subtle perturbations in their environment and prioritize pathways for human health assessment. Using an in vitro 3-D human full thickness skin model, we have examined the temporal response of dermal and epidermal layers to 10 cGy X-ray using transcriptomic, proteomic, phosphoproteomic and metabolomic platforms. Bioinformatics analysis of each dataset independently revealed potential signaling mechanisms affected by low dose radiation, and integrating data shed additional insight into the mechanisms regulating low dose responses in human tissue. We examined direct interactions among datasets (top down approach) and defined several hubs as significant regulators, including transcription factors (YY1, MYC and CREB1), kinases (CDK2, PLK1) and a protease (MMP2). These data indicate a shift in response across time — with an increase in DNA repair, tissue remodeling and repression of cell proliferation acutely (24–72 h). Pathway-based integration (bottom up approach) identified common molecular and pathway responses to low dose radiation, including oxidative stress, nitric oxide signaling and transcriptional regulation through the SP1 factor that would not have been identified by the individual data sets. Significant regulation of key downstream metabolites of nitrative stress was measured within these pathways. Among the features identified in our study, the regulation of MMP2 and SP1 was experimentally validated. Our results demonstrate the advantage of data integration to broadly define the pathways and networks that represent the mechanisms by which complex biological systems respond to perturbation. - Highlights: • Low dose ionizing radiation altered homeostasis in 3D skin tissue model. • Global gene/protein/metabolite data integrated using complementary statistical approaches • Time and location-specific change in matrix regulation, ROS/RNS and DNA repair pathways detected • Low dose exposure alters metabolites involved in nitric oxide biosynthesis and wound healing. • Computationally predicted regulators of primary mechanisms were experimentally validated.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Machine Learning of Bacterial Transcriptomes Reveals Responses Underlying Differential Antibiotic Susceptibility

In vitro antibiotic susceptibility testing often fails to accurately predict in vivo drug efficacies, in part due to differences in the molecular composition between standardized bacteriologic media and physiological environments within the body. Here, we investigate the interrelationship between antibiotic susceptibility and medium composition in Escherichia coli K-12 MG1655 as contextualized through machine learning of transcriptomics data. Application of independent component analysis, a signal separation algorithm, shows that complex phenotypic changes induced by environmental conditions or antibiotic treatment are directly traced to the action of a few key transcriptional regulators, including RpoS, Fur, and Fnr. Integrating machine learning results with biochemical knowledge of transcription factor activation reveals medium-dependent shifts in respiration and iron availability that drive differential antibiotic susceptibility. By extension, the data generation and data analytics workflow used here can interrogate the regulatory state of a pathogen under any measured condition and can be applied to any strain or organism for which sufficient transcriptomics data are available.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2014

Radiation Quality Effects on Transcriptome Profiles in 3-d Cultures After Particle Irradiation

In this work, we evaluate the differential effects of low- and high-LET radiation on 3-D organotypic cultures in order to investigate radiation quality impacts on gene expression and cellular responses. Reducing uncertainties in current risk models requires new knowledge on the fundamental differences in biological responses (the so-called radiation quality effects) triggered by heavy ion particle radiation versus low-LET radiation associated with Earth-based exposures. We are utilizing novel 3-D organotypic human tissue models that provide a format for study of human cells within a realistic tissue framework, thereby bridging the gap between 2-D monolayer culture and animal models for risk extrapolation to humans. To identify biological pathway signatures unique to heavy ion particle exposure, functional gene set enrichment analysis (GSEA) was used with whole transcriptome profiling. GSEA has been used extensively as a method to garner biological information in a variety of model systems but has not been commonly used to analyze radiation effects. It is a powerful approach for assessing the functional significance of radiation quality-dependent changes from datasets where the changes are subtle but broad, and where single gene based analysis using rankings of fold-change may not reveal important biological information. We identified 45 statistically significant gene sets at 0.05 q-value cutoff, including 14 gene sets common to gamma and titanium irradiation, 19 gene sets specific to gamma irradiation, and 12 titanium-specific gene sets. Common gene sets largely align with DNA damage, cell cycle, early immune response, and inflammatory cytokine pathway activation. The top gene set enriched for the gamma- and titanium-irradiated samples involved KRAS pathway activation and genes activated in TNF-treated cells, respectively. Another difference noted for the high-LET samples was an apparent enrichment in gene sets involved in cycle cycle/mitotic control. It is plausible that the enrichment in these particular pathways results from the complex DNA damage resulting from high-LET exposure where repair processes are not completed during the same time scale as the less complex damage resulting from low-LET radiation.

Patel, Z. S.↗