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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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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

The Importance of Protein Phosphorylation for Signaling and Metabolism in Response to Diel Light Cycling and Nutrient Availability in a Marine Diatom

Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and its role in regulation and signaling. We report a total of 2759 phosphorylation sites on 1502 proteins detected in Phaeodactylum tricornutum. Conditionally phosphorylated peptides were detected at low iron (n = 108), during the diel cycle (n = 149), and due to nitrogen availability (n = 137). Through a multi-omic comparison of transcript, protein, phosphorylation, and protein homology, we identify numerous proteins and key cellular processes that are likely under control of phospho-regulation. We show that phosphorylation regulates: (1) carbon retrenchment and reallocation during growth under low iron, (2) carbon flux towards lipid biosynthesis after the lights turn on, (3) coordination of transcription and translation over the diel cycle and (4) in response to nitrogen depletion. We also uncover phosphorylation sites for proteins that play major roles in diatom Fe sensing and utilization, including flavodoxin and phytotransferrin (ISIP2A), as well as identify phospho-regulated stress proteins and kinases. These findings provide much needed insight into the roles of protein phosphorylation in diel cycling and nutrient sensing in diatoms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Use of virus‐induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize

Abstract Plant disease resistance proteins (R‐proteins) detect specific pathogen‐derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1‐D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1‐D21 ‐induced HR. Here we describe a system for characterizing genes mediating HR, using virus‐induced gene silencing (VIGS) in a maize line carrying Rp1‐D21 . We assess the roles of 12 candidate genes. Three of these genes, SGT1 , RAR1 , and HSP90 , are required for HR induced by a number of R‐proteins across several plant–pathogen systems. We confirmed that maize HSP90 was required for full Rp1‐D21 ‐induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1‐D21 ‐induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O‐methyltransferase . We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein ( IQM3 ) and vacuolar protein sorting protein 37 , suppressed Rp1‐D21 ‐induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Quantitative Proteomics and Quantitative PCR as Predictors of cis -1,2-Dichlorethene and Vinyl Chloride Reductive Dechlorination Rates in Bioaugmented Aquifer Microcosms

Quantitative measurement of process-specific biomarker genes of Dehalococcoides mccartyi (Dhc) supports monitoring at chlorinated ethene contaminated sites. In this study, we varied Dhc cell abundances from ~103 to 108 cells/mL in aquifer microcosms and correlated the corresponding reductive dehalogenase (RDase) gene and RDase protein abundances with measured reductive dechlorination (RD) rates of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC). An additional set of microcosms tested the RD rate-predictive power of the regression analyses. These efforts revealed (1) that targeted proteomics quantifies Dhc biomarker proteins (e.g., TceA and VcrA, OmeA) over a relevant range of Dhc cell densities, and (2) that protein and gene abundances can predict RD rates. Protein detection limits translated to a rate coefficient of 10 –4 day –1 (0.04 year –1 ) for both k cDCE and k vc , which is within the range observed at sites undergoing monitored natural attenuation (MNA) (i.e., without the implementation of enhanced bioremediation treatment). Rates predicted using a combination of quantitative biomarker gene and protein measurements generally resulted in the best match with experimentally determined rate constants. These new findings provide evidence that quantitative biomarker measurements may be useful predictors of in situ RD rates, which would constitute a major advance for the cost-effective management of contaminated sites.

42 ENGINEERING↗
DOE OSTI2022

The development of extracellular vesicle markers for the fungal phytopathogen Colletotrichum higginsianum

Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14-3-3 protein (Bmh1) as potential EV markers and generated transgenic strains expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV interkingdom communication between plants and fungi.

14-3-3 proteins↗
DOE OSTI2023

Protein remote homology detection and structural alignment using deep learning

Exploiting sequence–structure–function relationships in biotechnology requires improved methods for aligning proteins that have low sequence similarity to previously annotated proteins. We develop two deep learning methods to address this gap, TM-Vec and DeepBLAST. TM-Vec allows searching for structure–structure similarities in large sequence databases. It is trained to accurately predict TM-scores as a metric of structural similarity directly from sequence pairs without the need for intermediate computation or solution of structures. Once structurally similar proteins have been identified, DeepBLAST can structurally align proteins using only sequence information by identifying structurally homologous regions between proteins. It outperforms traditional sequence alignment methods and performs similarly to structure-based alignment methods. We show the merits of TM-Vec and DeepBLAST on a variety of datasets, including better identification of remotely homologous proteins compared with state-of-the-art sequence alignment and structure prediction methods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

AI-accelerated protein-ligand docking for SARS-CoV-2 is 100-fold faster with no significant change in detection

Protein-ligand docking is a computational method for identifying drug leads. The method is capable of narrowing a vast library of compounds down to a tractable size for downstream simulation or experimental testing and is widely used in drug discovery. While there has been progress in accelerating scoring of compounds with artificial intelligence, few works have bridged these successes back to the virtual screening community in terms of utility and forward-looking development. We demonstrate the power of high-speed ML models by scoring 1 billion molecules in under a day (50 k predictions per GPU seconds). We showcase a workflow for docking utilizing surrogate AI-based models as a pre-filter to a standard docking workflow. Our workflow is ten times faster at screening a library of compounds than the standard technique, with an error rate less than 0.01% of detecting the underlying best scoring 0.1% of compounds. Our analysis of the speedup explains that another order of magnitude speedup must come from model accuracy rather than computing speed. In order to drive another order of magnitude of acceleration, we share a benchmark dataset consisting of 200 million 3D complex structures and 2D structure scores across a consistent set of 13 million “in-stock” molecules over 15 receptors, or binding sites, across the SARS-CoV-2 proteome. We believe this is strong evidence for the community to begin focusing on improving the accuracy of surrogate models to improve the ability to screen massive compound libraries 100 × or even 1000 × faster than current techniques and reduce missing top hits. The technique outlined aims to be a fast drop-in replacement for docking for screening billion-scale molecular libraries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. In this paper, we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that include neither the C-terminus nor the N-terminus of the protein. Conventionally, internal fragments have been disregarded, as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ~20–40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ~50 to ~99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. When searching for internal fragments during data analysis, previously unassigned peaks can be readily and accurately assigned to confirm a given protein sequence and to enhance the utility of top-down protein sequencing experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

In-Depth Proteome Profiling of Small Extracellular Vesicles Isolated from Cancer Cell Lines and Patient Serum

Extracellular Vesicle (EV) secretion has been observed from most types of normal and tumor cells. These EVs contain a variety of distinctive cargo where tumor-derived serum proteins in EVs secreted into the bloodstream can be used to provide a minimally invasive method for clinical monitoring. We have therefore undertaken a comprehensive study of the protein content of the EVs from several cancer cell lines using direct Data Independent Analysis (DIA). Further, several thousand proteins were detected including many of the classic EV markers such as CD9, CD81, CD63, TSG101 and Syndecan-1 among others. Many distinctive cancer related proteins were detected and some of them are known to be potential markers of cancer detection and monitoring of cancer progression. We have further studied the protein content of EVs from patient serum for both normal controls and pancreatic (PC) and Hepatocellular Carcinoma (HCC) cancers. The EVs for these studies have been isolated by various methods for comparison including ultracentrifugation (UC) and CD9 immunoaffinity column (CD9-IC). Typically, 500-1,000 proteins were identified where most of them overlapped with the EV proteins identified from the cell lines studied. Many of the EV protein markers were identified in these serum EVs and large numbers of proteins related to pancreatic and HCC cancers were identified.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Use of Fluorescent Protein Reporters for Assessing and Detecting Genome Editing Reagents and Transgene Expression in Plants

Fluorescent protein reporters have been widely used for monitoring the expression of target genes in various engineered organisms. Although a wide range of analytical approaches (e.g., genotyping PCR, digital PCR, DNA sequencing) have been utilized to detect and identify genome editing reagents and transgene expression in genetically modified plants, these methods are usually limited to use in the late stages of plant transformation and can only be used invasively. Here we describe GFP- and eYGFPuv-based strategies and methods for assessing and detecting genome editing reagents and transgene expression in plants, including protoplast transformation, leaf infiltration, and stable transformation. These methods and strategies enable easy, noninvasive screening of genome editing and transgenic events in plants.

Yuan, Guoliang↗
DOE OSTI2021

Protein and nucleic acid detection for microfluidic devices

The present invention relates to methods for detecting targets by employing a temperature control system with a microfluidic device. The system allows for non-contact heating by employing an infrared emitter. In some instances, the system can be used in conjunction with a centrifugal microfluidic device. Optionally, a mask can be implemented to provide selective heating of desired assay areas of the device.

Koh, Chung-Yan↗
DOE OSTI2020

Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria ( Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (C i ), but some evidence indicates that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of C i incorporation into different taxa, mat samples were incubated with [ 13 C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of 13 C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with 13 C. Most 13 C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins associated with photosynthesis, C i fixation, and N 2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for C i fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to C i fixation. Other taxa showed much lower [ 13 C]bicarbonate incorporation. IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven C i fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in C i fixation. Because this pathway imparts a lower degree of selection of isotopically heavy C i than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of 13 C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the 13 C/ 12 C ratios of mat biomass will help geochemists interpret the 13 C/ 12 C ratios of organic carbon in the fossil record.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

BAP1 antagonizes WWP1-mediated transcription factor KLF5 ubiquitination and inhibits autophagy to promote melanoma progression

Accumulating evidence revealed the abnormal expression of KLF5 in human cancers while its role in melanoma remains uncharacterized. This study aimed to explore the role of KLF5 in the proliferation and metastasis of melanoma. Bioinformatics analysis was performed to detect WWP1, BAP1 and KLF5 expression in melanoma, followed by expression determination on clinical tissues from melanoma patients and cancer cells. The cancer cells were infected with lentivirus expressing KLF5 or BAP1 while PI3K, AKT and mTOR expression was detected and autophagy was observed. Treated cells were injected to mice when tumor growth was measured and autophagy-related protein was detected. Plasmids expressing WWP1 and Ub-K48 were co-transfected into treated melanoma cells while immunoprecipitation assay was performed to determine the interaction among KLF5, WWP1, and BAP1. WWP1 was poorly expressed in melanoma cells and tissues whereas KLF5 was highly expressed and was positively correlated to poor prognosis. KLF5 promoted melanoma cell malignant phenotypes as well as inhibited autophagy. Interestingly, KLF5 contributed to activation of PI3K-AKT-mTOR signaling pathway, thereby inhibiting autophagy in melanoma cells. WWP1 mediated K48-linked ubiquitination of KLF5 to promote its degradation, and BAP1 antagonized this modification and stabilized KLF5 protein expression. Besides, BAP1 promoted KLF5-mediated growth of melanoma in vivo. Taken altogether, BAP1 antagonized WWP1-mediated ubiquitination of KLF5 to inhibit autophagy and promote melanoma development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Transcription of hepatitis B surface antigen shifts from cccDNA to integrated HBV DNA during treatment

The cornerstone of functional cure for chronic hepatitis B (CHB) is hepatitis B surface antigen (HBsAg) loss from blood. HBsAg is encoded by covalently closed circular DNA (cccDNA) and HBV DNA integrated into the host genome (iDNA). Nucleos(t)ide analogs (NUCs), the mainstay of CHB treatment, rarely lead to HBsAg loss, which we hypothesized was due to continued iDNA transcription despite decreased cccDNA transcription. To test this, we applied a multiplex droplet digital PCR that identifies the dominant source of HBsAg mRNAs to 3,436 single cells from paired liver biopsies obtained from 10 people with CHB and HIV receiving NUCs. With increased NUC duration, cells producing HBsAg mRNAs shifted their transcription from chiefly cccDNA to chiefly iDNA. This shift was due to both a reduction in the number of cccDNA-containing cells and diminished cccDNA-derived transcription per cell; furthermore, it correlated with reduced detection of proteins deriving from cccDNA but not iDNA. Despite this shift in the primary source of HBsAg, rare cells remained with detectable cccDNA-derived transcription, suggesting a source for maintaining the replication cycle. Functional cure must address both iDNA and residual cccDNA transcription. Further research is required to understand the significance of HBsAg when chiefly derived from iDNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Comparative Performance Evaluation of Large Language Models for Extracting Molecular Interactions and Pathway Knowledge

Understanding the interactions and regulatory relationships among biomolecules is essential for deciphering complex biological systems and elucidating the mechanisms behind diverse biological functions. Traditionally, the collection of such molecular interaction data has relied on expert curation, a process that is both time-consuming and labor-intensive. To address these limitations, this study explores the use of large language models (LLMs) to automate the genome-scale extraction of molecular interaction knowledge. Here, we evaluate the performance of various LLMs on key biological tasks, including the identification of protein-protein interactions, detection of genes associated with pathways influenced by low-dose radiation, and inference of gene regulatory relationships. Our findings demonstrate that larger LLMs tend to perform better, particularly in extracting intricate gene and protein interactions. Despite their strengths, these models face challenges in recognizing functionally diverse gene groups and highly correlated regulatory relationships. Through a comprehensive analysis using established molecular interaction and pathway databases, we show that LLMs possess the potential to identify relevant biomolecules and predict their interactions, offering valuable insights and marking a significant step toward AI-driven biological knowledge discovery.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗