Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “Expression”

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.

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

At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
NASA NTRSDate not supplied

AMO-EXPRESS-2.5: Crew Autonomy Onboard the International Space Station

NASA is committed to landing American astronauts, including the first woman and the next man, on the Moon by 2024. Currently, the crew cannot take on all functions performed by the ground today, so the future crews will need more automation to reduce the crew workload for future missions. Of significant importance for these missions is the balance between crew autonomy and vehicle automation. The Advanced Exploration Systems (AES) Autonomous Systems and Operations (ASO) Project has been investigating the ability to evaluate crew self-scheduling and activity monitoring for future space missions. The ASO project designed the Autonomous Mission and Operations- EXpedite the PRocessing of Experiments to Space Station Rack-2.5 (AMO-EXPRESS-2.5) payload to evaluate crew self-scheduling and activity monitoring. The AMO-EXPRESS-2.5 builds on the previous AMO-EXPRESS and AMO-EXPRESS-2.0 demonstrations on ISS. The AMO-EXPRESS-2.5 demonstration goals are to prove crew self-scheduling by planning through diagnosing systems expertise, failure detection, procedure recommendation and situational awareness. This paper will describe the development, test and execution results of the AMO-EXPRESS-2.5 demonstration, and will also outline the future planned development and operational efforts to enable autonomy for future deep space manned missions.

Brooke C. Allen↗
DOE OSTI2023

Cloning and expression vectors and systems

Vectors for cloning, maintaining and expressing a wide range of coding sequences in inducible T7 expression systems in Escherichia coli expression hosts are disclosed herein. Target genes that can be stably maintained and expressed include those that specify proteins that are highly toxic to the host cell. Different configurations of vectors and expression hosts provide different rates of transcription and translation of target genes and therefore different rates of accumulation of target proteins. Methods for cloning by asymmetric ligation and co-expression of more than one target protein in a single vector are also disclosed, as are variants of BL21(DE3) having lower basal transcription by T7 RNA polymerase.

Studier, F. William↗
DOE OSTI2020

Do Programmers Prefer Predictable Expressions in Code?

Source code is a form of human communication, albeit one where the information shared between the programmers reading and writing the code is constrained by the requirement that the code executes correctly. Programming languages are more syntactically constrained than natural languages, but they are also very expressive, allowing a great many different ways to express even very simple computations. Still, code written by developers is highly predictable, and many programming tools have taken advantage of this phenomenon, relying on language model surprisal as a guiding mechanism. Additionally, while surprisal has been validated as a measure of cognitive load in natural language, its relation to human cognitive processes in code is still poorly understood. In this paper, we explore the relationship between surprisal and programmer preference at a small granularity—do programmers prefer more predictable expressions in code? Using meaning-preserving transformations, we produce equivalent alternatives to developer-written code expressions and run a corpus study on Java and Python projects. In general, language models rate the code expressions developers choose to write as more predictable than these transformed alternatives. Then, we perform two human subject studies asking participants to choose between two equivalent snippets of Java code with different surprisal scores (one original and transformed). We find that programmers do prefer more predictable variants, and that stronger language models like the transformer align more often and more consistently with these preferences.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2024

Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression

ABSTRACT Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers’ deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus’s central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS’s function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion. IMPORTANCE Brown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of “omics” platforms has recently uncovered the key “oxidative-hydrolytic” mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes’ catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system’s use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Molecular Pathogenesis and Immune Evasion of Vesicular Stomatitis New Jersey Virus Inferred from Genes Expression Changes in Infected Porcine Macrophages

The molecular mechanisms associated with the pathogenesis of vesicular stomatitis virus (VSV) in livestock remain poorly understood. Several studies have highlighted the relevant role of macrophages in controlling the systemic dissemination of VSV during infection in different animal models, including mice, cattle, and pigs. To gain more insight into the molecular mechanisms used by VSV to impair the immune response in macrophages, we used microarrays to determine the transcriptomic changes produced by VSV infection in primary cultures of porcine macrophages. The results indicated that VSV infection induced the massive expression of multiple anorexic, pyrogenic, proinflammatory, and immunosuppressive genes. Overall, the interferon (IFN) response appeared to be suppressed, leading to the absence of stimulation of interferon-stimulated genes (ISG). Interestingly, VSV infection promoted the expression of several genes known to downregulate the expression of IFNβ. This represents an alternate mechanism for VSV control of the IFN response, beyond the recognized mechanisms mediated by the matrix protein. Although there was no significant differential gene expression in macrophages infected with a highly virulent epidemic strain compared to a less virulent endemic strain, the endemic strain consistently induced higher expression of all upregulated cytokines and chemokines. Collectively, this study provides novel insights into VSV molecular pathogenesis and immune evasion that warrant further investigation.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged approximately 35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

NASA Discipline Evolutionary Biology↗
NASA NTRS2002

AmphiFoxE4, an amphioxus winged helix/forkhead gene encoding a protein closely related to vertebrate thyroid transcription factor-2: expression during pharyngeal development

The full-length sequence and developmental expression of amphioxus AmphiFoxE4 are described. Transcripts of the gene are first detected in the pharyngeal endoderm, where the club-shaped gland is forming and subsequently in the definitive gland itself. AmphiFoxE4 is closely related to vertebrate genes encoding the thyroid-specific transcription factor-2 (TTF2), which plays an early developmental role in the morphogenesis of the thyroid gland and a later role in hormone-mediated control of thyroid function. In amphioxus, AmphiFoxE4 expression is not thyroid specific because the club-shaped gland, the only structure expressing the gene, is not homologous to the vertebrate thyroid; instead, the thyroid homologue of amphioxus is a specialized region of the pharyngeal endoderm called the endostyle. We propose that (a) the pharynx of an amphioxus-like ancestor of the vertebrates included a club-shaped gland that expressed FoxE4 as well as an endostyle that did not, and (b) the club-shaped gland soon disappeared in the vertebrate line of descent but (c) not before there was a homeogenetic transfer of FoxE4 expression from the club-shaped gland to the nearby endostyle. Such a transfer could have provided part of the genetic program enabling the endostyle to separate from the pharyngeal endoderm and migrate away as the rudiment of the thyroid gland.

Non-NASA Center↗
NASA NTRS2001

Hindlimb unweighting decreases endothelium-dependent dilation and eNOS expression in soleus not gastrocnemius

We tested the hypothesis that hindlimb unweighting (HLU) decreases endothelium-dependent vasodilation and expression of endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) in arteries of skeletal muscle with reduced blood flow during HLU. Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 15) or control (n = 15) conditions for 14 days. ACh-induced dilation was assessed in muscle with reduced [soleus (Sol)] or unchanged [gastrocnemius (Gast)] blood flow during HLU. eNOS and SOD-1 expression were measured in feed arteries (FA) and in first-order (1A), second-order (2A), and third-order (3A) arterioles. Dilation to infusion of ACh in vivo was blunted in Sol but not Gast. In arteries of Sol muscle, HLU decreased eNOS mRNA and protein content. eNOS mRNA content was significantly less in Sol FA (35%), 1A arterioles (25%) and 2A arterioles (18%). eNOS protein content was less in Sol FA (64%) and 1A arterioles (65%) from HLU rats. In arteries of Gast, HLU did not decrease eNOS mRNA or protein. SOD-1 mRNA expression was less in Sol 2A arterioles (31%) and 3A arterioles (29%) of HLU rats. SOD-1 protein content was less in Sol FA (67%) but not arterioles. SOD-1 mRNA and protein content were not decreased in arteries from Gast. These data indicate that HLU decreases endothelium-dependent vasodilation, eNOS expression, and SOD-1 expression primarily in arteries of Sol muscle where blood flow is reduced during HLU.

NASA Discipline Cardiopulmonary↗
NASA NTRS1999

Nitric-oxide synthase is a mechanical signal transducer that modulates talin and vinculin expression

Mechanical stimuli can cause changes in muscle mass and structure which indicate that mechanisms exist for transducing mechanical stimuli into signals that influence gene expression. Myotendinous junctions show adaptations to modified muscle loading which suggest that these are transcriptionally distinct domains in muscle fibers that may experience local regulation of expression of structural proteins that are concentrated at these sites. Vinculin and talin are cytoskeletal proteins that are highly enriched at myotendinous junctions that we hypothesize to be subject to local transcriptional regulation. Our findings show that mechanical stimulation of muscle cells in vivo and in vitro causes an increase in the expression of vinculin and talin that is mediated by nitric oxide. Furthermore, nitric oxide-stimulated increases in vinculin and talin expression occur through a protein kinase G-dependent pathway and therefore differ from other mechanisms through which nitric oxide has been shown previously to modulate transcription. Analysis of vinculin mRNA distribution in mechanically stimulated muscle fibers shows that the mRNA is highly concentrated at myotendinous junctions, which supports the hypothesis that myotendinous junctions are distinct domains in which the expression of cytoskeletal proteins is modulated by mechanical stimuli through a nitric oxide and protein kinase G-dependent pathway.

NASA Discipline Musculoskeletal↗
NASA NTRS1997

COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

NASA Discipline Cell Biology↗
DOE OSTI2022

Identification of a pharmacological approach to reduce ACE2 expression and development of an in vitro COVID-19 viral entry model

Because of rapid emergence and circulation of the SARS-CoV-2 variants, especially Omicron which shows increased transmissibility and resistant to antibodies, there is an urgent need to develop novel therapeutic drugs to treat COVID-19. In this study we developed an in vitro cellular model to explore the regulation of ACE2 expression and its correlation with ACE2-mediated viral entry. We examined ACE2 expression in a variety of human cell lines, some of which are commonly used to study SARS-CoV-2. Using the developed model, we identified a number of inhibitors which reduced ACE2 protein expression. The greatest reduction of ACE2 expression was observed when CK869, an inhibitor of the actin-related protein 2/3 (ARP2/3) complex, was combined with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of sodium-hydrogen exchangers (NHEs), after treatment for 24 h. Using pseudotyped lentivirus expressing the SARS-CoV-2 full-length spike protein, we found that ACE2-dependent viral entry was inhibited in CK869 + EIPA-treated Calu-3 and MDA-MB-468 cells. This study provides an in vitro model that can be used for the screening of novel therapeutic candidates that may be warranted for further pre-clinical and clinical studies on COVID-19 countermeasures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Maternal undernutrition modulates hepatic MicroRNAs expression in the early life of offspring

Emerging studies revealed that a poor intrauterine environment elicited by maternal nutrient restriction (MNR) is associated with an increased risk of metabolic diseases in adulthood. Previous research has shown that microRNAs (miRNAs) exert pivotal roles in modulating molecular pathways involved in disease pathogenesis and progression. In this respect, we herein examined miRNA profiles in samples of liver from offspring whose mothers were fed either with a 50% food-restricted diet or standard laboratory chow during pregnancy. Our findings enumerated that miR-181a, involved in lipid metabolism, was found to be downregulated in the liver of MNR offspring at 1 day of age when compared to that of control offspring. We also noted that overexpression of miR-181a reduced the lipid droplets after treatment with oleic acid for 48 h, which suppressed the expressions levels of SIRT1, FOXO1, KLF6 and PPARγ in BRL-3A cells, while the opposite results were observed with decreased expression of miR-181a. Furthermore, the luciferase reporter assay confirmed the direct interactions between miR-181a with KLF6 and SIRT1. In adults, the MNR offspring elucidated increased TG content, decreased expression of miR-181a, and increased expressions levels of SIRT1, FOXO1, KLF6, and PPARγ in liver tissues. Collectively, these findings provided novel evidence that MNR could regulate miRNAs expression, which might be related to lipid metabolism in MNR offspring.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Glucocorticoids suppressed osteoblast differentiation by decreasing Sema3A expression via the PIK3/Akt pathway

Highlights: • Dex decreased Sema3A expression by suppressing the PI3K/Akt pathway. • Sema3A reversed Dex-induced suppression of osteoblast differentiation. • Sema3A reversed the Dex-mediated reduced transcriptional activity of β-catenin. Glucocorticoids(GCs) are extensively used to treat inflammatory and autoimmune diseases. Excessive prolonged exposure to glucocorticoids is associated with an increased risk of osteoporosis. The inhibition of osteoblast differentiation by GCs is suggested as a major cause for GCs-induced osteoporosis (GIO). However, the precise mechanism underlying the role of GCs in osteoblasts differentiation is not fully elucidated. Semaphorin 3A (Sema3A), a secreted member of the Semaphorin family, enhances bone formation and promotes fracture healing, which is known to increase osteoblastic differentiation and stimulate osteogenesis in bone metabolism. Here, the present study explored the effect of Sema3A in osteoblast differentiation using dexamethasone (Dex) treatment of bone marrow stromal cells (BMSCs). Dex treatment decreased Sema3A expression in BMSCs in a dose-dependent manner. Moreover, Dex stimulation suppressed the differentiation of osteoblasts by reducing alkaline phosphatase (ALP) activity, osteoblastic marker genes expression and mineralization, but all of these effects were ameliorated by exogenous recombinant Sema3A administration. Furthermore, exogenous Sema3A administration reversed the Dex-mediated decrease in nuclear accumulation of β-catenin and β-catenin activity in BMSCs. Meanwhile, Dex was capable of simultaneously suppressing the phosphorylation of protein kinase B(Akt) and the expression level of Sema3A in BMSCs. These changes were significantly abolished by the PI3K/Akt agonist. These results suggest that Dex inhibits osteoblast differentiation by suppressing Sema3A expression via the PI3K/Akt pathway. These data provide new insights into the molecular mechanisms of Dex-induced osteoblast differentiation inhibition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Expression Patterns of Energy-Related Genes in Single Cells Uncover Key Isoforms and Enzymes That Gain Priority Under Nanoparticle-Induced Stress

Cellular responses to nanoparticles (NPs) have been largely studied in cell populations, providing averaged values that often misrepresent the true molecular processes that occur in the individual cell. To understand how a cell redistributes limited molecular resources to achieve optimal response and survival requires single-cell analysis. Here we applied multiplex single molecule-based fluorescence in situ hybridization (fliFISH) to quantify the expression of 10 genes simultaneously in individual intact cells, including glycolysis and glucose transporter genes, which are critical for restoring and maintaining energy balance. We focused on individual gill epithelial cell responses to lithium cobalt oxide (LCO) NPs, which are actively pursued as cathode materials in lithium-ion batteries, raising concerns about their impact on the environment and human health. We found large variabilities in the expression levels of all genes between neighboring cells under the same exposure conditions, from only a few transcripts to over 100 copies in individual cells. Gene expression ratios among the 10 genes in each cell uncovered shifts in favor of genes that play key roles in restoring and maintaining energy balance. Among these genes are isoforms that can secure and increase glycolysis rates more efficiently, as well as genes with multiple cellular functions, in addition to glycolysis, including DNA repair, regulation of gene expression, cell cycle progression, and proliferation. Our study uncovered prioritization of gene expression in individual cells for restoring energy balance under LCO NP exposures. Broadly, our study gained insight into single-cell strategies for redistributing limited resources to achieve optimal response and survival under stress.

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

Engineering Clostridium Tyrobutyricum for High Butanol Production through Induction Expression of Exogenous NADPH-Dependent HBD

Clostridium tyrobutyricum Δ cat 1:: adh E2 is a promising cell factory for butanol production because of its robustness, high butanol tolerance, and minimal butyrate production. However, excessive acetate and ethanol production remains a major bottleneck limiting its butanol yield. Coexpressing an exogenous hbd ( Ck ) encoding the NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase (HBD) from Clostridium kluyveri with adh E2 could increase the C4 carbon flux, resulting in increased butanol and decreased acetate and ethanol production. However, constitutively overexpressing hbd ( Ck ) in Δ cat 1:: adh E2 shows little improvement in butanol yield, productivity, and selectivity, which might be caused by redox imbalance and growth inhibition. To alleviate this problem, C. tyrobutyricum MΔ cat 1:: adh E2- Pbgal-hbd ( Ck ) with a dynamic expression of hbd ( Ck ) controlled by an inducible promoter was developed. In serum bottle fermentation at 37 °C, when the hbd ( Ck ) expression was induced at 12 h or in the early exponential phase, butanol production increased ∼20% in yield (from 0.22 to 0.27 g/g glucose), 87.5% in productivity (from 0.16 to 0.30 g/L·h), and 52% in selectivity (from 0.46 to 0.70 g/g total products) compared to the control strain without expressing any hbd(Ck), whereas hbd ( Ck ) expression induced at 0 or 24 h in MΔ cat 1:: adh E2-P bgal - hbd ( Ck ) or constitutively in MΔ cat 1:: adh E2-P cat 1- hbd ( Ck ) showed significantly lower butanol yield and productivity. At 25 °C, MΔ cat 1:: adh E2-P bgal - hbd ( Ck ) with 12 h induction produced the highest butanol titer of 23 g/L with 0.32 g/g yield, 0.16 g/L·h productivity, and 0.83 g/g product selectivity due to much reduced acetate formation. Subsequent scale-up to a stirred-tank bioreactor at 37 °C increased productivity to 0.39 g/L·h while also achieving high butanol titer (21.8 g/L), yield (0.30 g/g), and selectivity (0.67 g/g). The optimized induction timing resulted in a balanced NAD(P)H pool, effectively channeling substrates toward butanol biosynthesis. It was concluded that the timing for hbd ( Ck ) expression was critical as it affected glucose catabolism, cell growth, redox balance, and carbon flux distribution. These findings underscore the potential of dynamic metabolic regulation to overcome bottlenecks in biobutanol production, providing a scalable and economically viable bioprocess for industrial application.

Clostridium tyrobutyricum↗
DOE OSTI2021

A short hepatitis C virus NS5A peptide expression by AAV vector modulates human T cell activation and reduces vector immunogenicity

Viral vector-mediated gene therapies have the potential to treat many human diseases; however, host immune responses against the vector and/or the transgene pose a safety risk to the patients and can negatively impact product efficacy. Thus, novel strategies to reduce vector immunogenicity are critical for the advancement of these therapies. T cell activation (TCA) is required for the development of immune responses during gene therapy. We hypothesized that modulation of TCA by incorporating a novel viral immunomodulatory factor into a viral vector may reduce unwanted TCA and immune responses during gene therapy. To test this hypothesis, we identified an immunomodulatory domain of the hepatitis C virus (HCV) NS protein 5A (NS5A) protein and studied the effect of viral vectors expressing NS5A peptide on TCA. Lentiviral vector-mediated expression of a short 20-mer peptide derived from the NS5A protein in human T cells was sufficient to inhibit TCA. Synthetic 20-mer NS5A peptide also inhibited TCA in primary human T cells. Mechanistically, the NS5A protein interacted with Lck and inhibited proximal TCR signaling. Importantly, NS5A peptide expression did not cause global T cell signaling dysfunction as distal T cell signaling was not inhibited. Finally, recombinant adeno-associated virus (AAV) vector expressing the 20-mer NS5A peptide reduced both the recall antigen and the TCR-mediated activation of human T cells and did not cause global T cell signaling dysfunction. Together, these data suggest that expression of a 20-mer NS5A peptide by an AAV vector may reduce unwanted TCA and may contribute to lower vector immunogenicity during gene therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants

Abstract Green fluorescent protein (GFP) has been widely used for monitoring gene expression and protein localization in diverse organisms. However, highly sensitive imaging equipment, like fluorescence microscope, is usually required for the visualization of GFP, limitings its application to fixed locations in samples. A reporter that can be visualized in real-time regardless the shape, size and location of the target samples will increase the flexibility and efficiency of research work. Here, we report the application of a GFP-like protein, called eYGFPuv, in both transient expression and stable transformation, in two herbaceous plant species ( Arabidopsis and tobacco) and two woody plant species (poplar and citrus). We observed bright fluorescence under UV light in all of the four plant species without any effects on plant growth or development. eYGFPuv was shown to be effective for imaging transient expression in leaf and root tissues. With a focus on in vitro transformation, we demonstrated that the transgenic events expressing 1x eYGFPuv could be easily identified visually during the callus stage and the shoot stage, enabling early and efficient selection of transformants. Furthermore, whole-plant level visualization of eYGFPuv revealed its ubiquitous stability in transgenic plants. In addition, our transformation experiments showed that eYGFPuv can also be used to select transgenic plants without antibiotics. This work demonstrates the feasibility of utilizing 1x eYGFPuv in studies of gene expression and plant transformation in diverse plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Accurate flux predictions using tissue-specific gene expression in plant metabolic modeling

The accurate prediction of complex phenotypes such as metabolic fluxes in living systems is a grand challenge for systems biology and central to efficiently identifying biotechnological interventions that can address pressing industrial needs. The application of gene expression data to improve the accuracy of metabolic flux predictions using mechanistic modeling methods such as flux balance analysis (FBA) has not been previously demonstrated in multi-tissue systems, despite their biotechnological importance. We hypothesized that a method for generating metabolic flux predictions informed by relative expression levels between tissues would improve prediction accuracy. Relative gene expression levels derived from multiple transcriptomic and proteomic datasets were integrated into FBA predictions of a multi-tissue, diel model of Arabidopsis thaliana’s central metabolism. This integration dramatically improved the agreement of flux predictions with experimentally based flux maps from 13 C metabolic flux analysis compared with a standard parsimonious FBA approach. Disagreement between FBA predictions and MFA flux maps was measured using weighted averaged percent error values, and for parsimonious FBA this was 169%–180% for high light conditions and 94%–103% for low light conditions, depending on the gene expression dataset used. This fell to 10%-13% and 9%-11% upon incorporating expression data into the modeling process, which also substantially altered the predicted carbon and energy economy of the plant.

59 BASIC BIOLOGICAL SCIENCES↗