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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 OSTI2020

COnTORT: COmprehensive Transcriptomic ORganizational Tool for Simultaneously Retrieving and Organizing Numerous Gene Expression Data Sets from the NCBI Gene Expression Omnibus Database

We introduce COnTORT ( CO mprehensive T ranscriptomic OR ganizational T ool), a publicly available program that retrieves all available gene expression data and associated metadata for an organism from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. The data are compiled into text files that can be used for downstream bioinformatic applications.

Myers, Kevin S.↗
DOE OSTI2026

MassIVE MSV000100641 - Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli: Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

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Alexander, William G. [Oak Ridge National Laborato↗
DOE OSTI2026

Comparison of gene expression in the skin tissue of gray, humpback, and fin whales

Analyses of gene expression in the skin of several species of whales identified genes that are differentially expressed in association with environmental factors, suggesting that skin transcriptomics may provide a valuable tool for assessing physiological responses in marine mammals. Previous work exploring differing levels of gene expression has focused on odontocetes with comparatively limited investigation of skin gene expression has been explored in mysticetes. Here, we describe the identity of genes expressed in skin tissue of three species of baleen whales to establish a baseline of gene expression and compare gene identity and expression patterns across species. We also evaluate sex-specific differences in skin gene expression through a comparison of expression levels between males and females in gray and humpback whales. A total of 16 skin tissue samples were collected from free-ranging gray, humpback and fin whales off the central Oregon coast in the eastern North Pacific. Comparison of the expressed genes in the humpback and gray whale skin tissue to the blue whale reference database identified enriched gene ontology terms in the skin tissue of each species, suggesting genes over-represented in the whale skin related to cell epithelial development, regulation of gene expression and cell maintenance . Comparison of gene expression between male and female samples revealed sex-specific differences in gray and humpback whales. A differential gene expression analysis identified several x-linked genes that have been previously identified and show gene expression differences in male and female cetaceans, such as ZFX, DDX3X and USP9X. Establishing baseline skin gene expression profiles for these three baleen whale species sampled off the Oregon coast provides a foundation for linking transcriptome variation with physiological condition and environment.

Sremba, Angela↗
DOE OSTI2021

MiR-205–5p suppresses angiogenesis in gastric cancer by downregulating the expression of VEGFA and FGF1

Highlights: • MiR-205-5p was commonly downregulated in GC tissues from TCGA database and our hospital, and these events were associated with a worse TNM stagedistant metastasis and poor survival. • MiR-205-5p and CD31 expressions were negatively inter-related within GC tissues from TCGA database and our hospital. • GC cell lines expressed lower level of miR-205-5p significantly as compared with the normal gastric cell line. • MiR-205-5p upregulation significantly impaired proliferation and colony formation of GC cells and neovascularization induced by GC cells in vitro and in vivo. • The angiogenesis and ERK-signaling were depressed by VEGFA and FGF1 downregulation induced by miR-205-5p overexpression. The miR-205-5p inhibitor promoted malignant phenotypes by enhancing VEGFA and FGF1 expressions, as well as the activation of ERK-signaling. Anti-angiogenic therapy represents one of the most promising treatment modalities for human cancers. However, the response to antiangiogenic therapy in gastric cancer (GC) remains dismal. To help identify new strategies for antiangiogenic therapy in GC, we evaluated miR-205–5p expression in GC tissues from TCGA database and our hospital, and its functions in angiogenesis were explored in vitro and in vivo. We investigated miR-205–5p expression and microvessel densities (MVDs) in GC tissues and liver metastases from patients. The function and mechanisms of miR-205–5p were examined in human cell lines and in xenograft mouse models. Associations between miR-205–5p expression and clinical characteristics were analyzed using either Pearson's χ{sup 2} test or Fisher's exact test. Differences in overall survival (OS) distributions were evaluated using the log-rank test. Differences in measurement data were compared using Student's t-test and one-way ANOVA. We found that miR-205–5p expression was downregulated in GC tissues and was negatively correlated with CD31 expression in both TCGA and our clinical samples. GC cell lines expressed low levels of miR-205–5p, and miR-205–5p upregulation significantly impaired the proliferation and angiogenesis of GC cells. Moreover, vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 1 (FGF1) expression and activation of extracellular-related kinase (ERK) signaling were suppressed by miR-205–5p. MiR-205–5p inhibition promoted malignant phenotypes by enhancing VEGFA and FGF1 expression, as well as the activation of ERK signaling. Angiogenesis and ERK signaling were decreased in response to VEGFA and FGF1 downregulation induced by miR-205–5p overexpression. The dual-luciferase reporter assay showed that VEGFA and FGF1 were direct targets of miR-205–5p. Xenograft mouse models revealed that miR-205–5p suppressed tumor growth by inhibiting neovascularization. Altogether, these results demonstrate that miR-205–5p suppresses angiogenesis in GC by attenuating the expression of VEGFA and FGF1, indicating that upregulation of miR-205–5p may represent as an antiangiogenic therapy for GC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Small Signaling Peptides in Sorghum bicolor : Integrating Phylogeny and Gene Expression to Characterize Roles in Stem Development

Small signaling peptides (SSPs) are important regulators of plant growth, development, and responses to biotic and abiotic stress, yet their role in the C4 grass Sorghum bicolor is largely uncharacterized. To help fill this knowledge gap, 219 sorghum genes that encode SSPs were identified based on SSP sequences previously identified in Arabidopsis thaliana, Zea mays, Oryza sativa, Triticum aestivum , and Brachypodium distachyon . The 219 sorghum SSP-encoding genes were assigned to 19 gene families, analyzed for the presence of motifs, and aligned with genes that encode SSPs in other plants using phylogenetic analysis. Sorghum genes in 12 of the 19 SSP gene families had not been previously characterized. Expression of the 219 SSP-encoding genes in sorghum organs, during stem development, and in stem tissues and cell types revealed distinct spatial, temporal, and developmental patterns of expression. Genes associated with the SbCEP and SbRGF families were preferentially expressed in roots, whereas SbEPF genes were expressed in stem epidermal and pith parenchyma cells and panicles. The expression of genes during bioenergy sorghum stem growth and development was investigated because stems account for ~80% of harvested biomass and serve as conduits for water and nutrient transport between leaves and roots. During stem development, 28 SSP genes in several families ( CLE, EPF, CEP, GASS, PSY, ES, PSK, CAPE, POE ) were expressed at higher levels in zones of cell proliferation. For example, the TDIF homologs SbCLE41 and SbCLE42 were expressed at high levels in nascent stem nodes where they may regulate vascular bundle cambial activity and cell differentiation. A different set of 15 genes in the CIF, POE, CAPE, PSY, CEP, RALF , and CLE families were expressed at higher levels in zones of stem tissue differentiation highlighted by elevated expression of five SbRALFR s in the stem nodal plexus. Cell type–specific expression of many sorghum genes that encode SSPs was observed in fully elongated internodes indicating gene expression is regulated with high spatial resolution. Overall, the results provide a foundation of information for analysis of SSP function in sorghum that can be integrated with knowledge of sorghum gene regulatory networks to modulate traits important for production of sorghum crops.

bioenergy sorghum↗
DOE OSTI2022

Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome

Eukaryotic green microalgae represent a sustainable, photosynthetic biotechnology platform for generating high-value products. The model green alga Chlamydomonas reinhardtii has already been used to generate high value bioproducts such as recombinant proteins and terpenoids. However, low, unstable, and variable nuclear transgene expression has limited the ease and speed of metabolic engineering and recombinant protein expression in this system. Here, novel genetic devices for transgene expression in C. reinhardtii have been developed by identifying cis-regulatory DNA elements capable of driving high transgene expression in C. reinhardtii promoters using de novo motif discovery informatics approaches. Thirteen putative motifs were synthesized as concatemers, linked to a common minimal basal promoter, and assayed for their activity to drive expression of a yellow fluorescent protein reporter gene. Following transformation of the vectors into C. reinhardtii by electroporation, in vivo measurements of yellow fluorescent protein expression by flow cytometry revealed that five of the DNA motifs analyzed displayed significantly higher reporter expression compared to the basal promoter control. Two of the concatemerized motifs, despite being much smaller minimal cis-regulatory elements, drove reporter expression at levels approaching that of the conventionally-used AR1 promoter. This analysis provides insight into C. reinhardtii promoter structure and gene regulation, and provides a new toolbox of cis-regulatory elements that can be used to drive transgene expression at a variety of expression levels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Evolutionary Analyses of Gene Expression Divergence in Panicum hallii : Exploring Constitutive and Plastic Responses Using Reciprocal Transplants

Abstract The evolution of gene expression is thought to be an important mechanism of local adaptation and ecological speciation. Gene expression divergence occurs through the evolution of cis- polymorphisms and through more widespread effects driven by trans-regulatory factors. Here, we explore expression and sequence divergence in a large sample of Panicum hallii accessions encompassing the species range using a reciprocal transplantation experiment. We observed widespread genotype and transplant site drivers of expression divergence, with a limited number of genes exhibiting genotype-by-site interactions. We used a modified FST–QST outlier approach (QPC analysis) to detect local adaptation. We identified 514 genes with constitutive expression divergence above and beyond the levels expected under neutral processes. However, no plastic expression responses met our multiple testing correction as QPC outliers. Constitutive QPC outlier genes were involved in a number of developmental processes and responses to abiotic environments. Leveraging earlier expression quantitative trait loci results, we found a strong enrichment of expression divergence, including for QPC outliers, in genes previously identified with cis and cis–environment interactions but found no patterns related to trans-factors. Population genetic analyses detected elevated sequence divergence of promoters and coding sequence of constitutive expression outliers but little evidence for positive selection on these proteins. Our results are consistent with a hypothesis of cis-regulatory divergence as a primary driver of expression divergence in P. hallii.

3′ TagSeq↗
DOE OSTI2023

A Synthetic Transcription Factor and Core Promoter System in Picochlorum renovo Enables Tunable Gene Expression

Picochlorum renovo is a recently characterized microalga of industrial interest. Its rapid growth rate, and high temperature and salinity tolerances make P. renovo an attractive candidate for industrial scale cultivation and downstream production of sustainable fuels and chemicals. Currently, genetic tools for many non-model microalgae are limited and would greatly benefit from an orthogonal gene expression system to bypass host regulation. Additionally, the engineering of complex metabolic pathways in eukaryotic organisms to optimize growth or biosynthesize high value products often requires tunable expression of each gene in a pathway. Here we explore a tunable orthogonal gene expression system using a synthetic transcription factor (sTF) and core promoters (CPs) conferring expression of the fluorescent protein mCherry to quantify protein expression. The sTF paired with the relevant binding site (BS) led to an ~5X increase in reporter gene expression compared to the native RuBisCo promoter, however had limited tunability with increasing BS number. Quantification of mCherry expression under 34 different CPs paired with the sTF and BS showed an order of magnitude of expression tunability. Future work with this system will entail generation of an overexpression library via random integration of the relevant BS in an sTF expressing P. renovo strain. With this sTF and CP system we aim to greatly improve growth rates and product titers in photosynthetic organisms, while also providing a potentially universal gene expression system for microalgae.

algae↗
DOE OSTI2021

Expression of membrane protein disulphide isomerase A1 (PDIA1) disrupt a reducing microenvironment in endometrial epithelium for embryo implantation

Various proteins in the endometrial epithelium are differentially expressed in the receptive phase and play a pivotal role in embryo implantation. The Protein Disulphide Isomerase (PDI) family contains 21 members that function as chaperone proteins through their redox activities. Although total PDIA1 protein expression was high in four common receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cell lines, significantly higher membrane PDIA1 expression was found in non-receptive AN3CA cells. In Ishikawa cells, oestrogen up-regulated while progesterone down-regulated membrane PDIA1 expression. Moreover, mid-luteal phase hormone treatment down-regulated membrane PDIA1 expression. Furthermore, oestrogen at 10 nM reduced spheroid attachment on Ishikawa cells. Interestingly, inhibition of PDIA1 function by bacitracin or 16F16 increased the spheroid attachment rate onto non-receptive AN3CA cells. Over-expression of PDIA1 in receptive Ishikawa cells reduced the spheroid attachment rate and significantly down-regulated integrin β3 levels, but not integrin αV and E-cadherin. Addition of reducing agent TCEP induced a sulphydryl-rich microenvironment and increased spheroid attachment onto AN3CA cells and human primary endometrial epithelial cells collected at LH+7/8 days. The luminal epithelial cells from human endometrial biopsies had higher PDIA1 protein expression in the proliferative phase than in the secretory phase. Our findings suggest oestrogen and progesterone regulate PDIA1 expression, resulting in the differential expressions of membrane PDIA1 protein to modulate endometrial receptivity. This suggests that membrane PDIA1 expression prior to embryo transfer could be used to predict endometrial receptivity and embryo implantation in women undergoing assisted reproduction treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system

Abstract Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli . Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus , RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

59 BASIC BIOLOGICAL SCIENCES↗