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At least 1,171 records · Page 65
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Mycorrhiza‐induced mycocypins of Laccaria bicolor are potent protease inhibitors with nematotoxic and collembola antifeedant activity

Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Finally, our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A chloroplast diacylglycerol lipase modulates glycerolipid pathway balance in Arabidopsis

SUMMARY Two parallel pathways compartmentalized in the chloroplast and the endoplasmic reticulum contribute to thylakoid lipid synthesis in plants, but how these two pathways are coordinated during thylakoid biogenesis and remodeling remains unknown. We report here the molecular characterization of a homologous ADIPOSE TRIGLYCERIDE LIPASE‐LIKE gene, previously referred to as ATGLL . The ATGLL gene is ubiquitously expressed throughout development and rapidly upregulated in response to a wide range of environmental cues. We show that ATGLL is a chloroplast non‐regioselective lipase with a hydrolytic activity preferentially towards 16:0 of diacylglycerol (DAG). Comprehensive lipid profiling and radiotracer labeling studies revealed a negative correlation of ATGLL expression and the relative contribution of the chloroplast lipid pathway to thylakoid lipid biosynthesis. Additionally, we show that genetic manipulation of ATGLL expression resulted in changes in triacylglycerol levels in leaves. We propose that ATGLL, through affecting the level of prokaryotic DAG in the chloroplast, plays important roles in balancing the two glycerolipid pathways and in maintaining lipid homeostasis in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Hidden diversity: Transcriptomic and photosynthetic variation among common ‘wild type’ Chlamydomonas strains

The unicellular green alga Chlamydomonas reinhardtii is a widely studied reference organism, particularly in photosynthesis research. It employs photoprotective mechanisms, such as state transitions (ST) and non-photochemical quenching (NPQ), to cope with rapid light changes. Most widely used strains share a recent common ancestor yet differ by up to ~50 000 nuclear variants—genetic diversity that is often overlooked. Even among ‘wild type’ strains, we document significant phenotypic differences, such as pigment accumulation, and nutrient utilization. To elucidate the basis for this variation, we compared transcriptomes and physiological traits of seven commonly used laboratory strains, including the reference strain and the CLiP mutant library parental strain. Despite identical growth conditions, ~40% of genes were differentially expressed between strains. Most of these differences are attributable to changes that have accrued during laboratory propagation, and adverse conditions may have driven transcriptomic drift. At the physiological level, we catalog the range of strain-dependent responses related to photosynthesis and high light (HL) acclimation. Specifically, (i) all strains develop NPQ upon HL exposure, but to various degrees, (ii) they show a substantial variation in ST capacity, and (iii) they regulate the composition of the photosynthetic apparatus differently. We find that NPQ levels do not correlate with LHCSR3 expression, suggesting an additional layer of NPQ regulation. STs are constantly activated and independent of growth light intensities. Overall, our findings highlight significant strain-to-strain differences in virtually all photosynthetic parameters, emphasizing the importance of careful strain selection in future research endeavors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A Melanoma Brain Metastasis CTC Signature and CTC:B-cell Clusters Associate with Secondary Liver Metastasis: A Melanoma Brain–Liver Metastasis Axis

Melanoma brain metastasis is linked to dismal prognosis and low overall survival and is detected in up to 80% of patients at autopsy. Circulating tumor cells (CTC) are the smallest functional units of cancer and precursors of fatal metastasis. We previously used an unbiased multilevel approach to discover a unique ribosomal protein large/small subunit (RPL/RPS) CTC gene signature associated with melanoma brain metastasis. In this study, we hypothesized that CTC-driven melanoma brain metastasis secondary metastasis (“metastasis of metastasis” per clinical scenarios) has targeted organ specificity for the liver. We injected parallel cohorts of immunodeficient and newly developed humanized NBSGW (huNBSGW) mice with cells from CTC-derived melanoma brain metastasis to identify secondary metastatic patterns. We found the presence of a melanoma brain–liver metastasis axis in huNBSGW mice. Furthermore, RNA sequencing analysis of tissues showed a significant upregulation of the RPL/RPS CTC gene signature linked to metastatic spread to the liver. Additional RNA sequencing of CTCs from huNBSGW blood revealed extensive CTC clustering with human B cells in these mice. CTC:B-cell clusters were also upregulated in the blood of patients with primary melanoma and maintained either in CTC-driven melanoma brain metastasis or melanoma brain metastasis CTC–derived cells promoting liver metastasis. CTC-generated tumor tissues were interrogated at single-cell gene and protein expression levels (10x Genomics Xenium and HALO spatial biology platforms, respectively). Collectively, our findings suggest that heterotypic CTC:B-cell interactions can be critical at multiple stages of metastasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structures of ISCth4 transpososomes reveal the role of asymmetry in copy-out/paste-in DNA transposition

Copy-out/paste-in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Machine and Deep Learning: Artificial Intelligence Application in Biotic and Abiotic Stress Management in Plants

Biotic and abiotic stresses significantly affect plant fitness, resulting in a serious loss in food production. Biotic and abiotic stresses predominantly affect metabolite biosynthesis, gene and protein expression, and genome variations. However, light doses of stress result in the production of positive attributes in crops, like tolerance to stress and biosynthesis of metabolites, called hormesis. Advancement in artificial intelligence (AI) has enabled the development of high-throughput gadgets such as high-resolution imagery sensors and robotic aerial vehicles, i.e., satellites and unmanned aerial vehicles (UAV), to overcome biotic and abiotic stresses. These High throughput (HTP) gadgets produce accurate but big amounts of data. Significant datasets such as transportable array for remotely sensed agriculture and phenotyping reference platform (TERRA-REF) have been developed to forecast abiotic stresses and early detection of biotic stresses. For accurately measuring the model plant stress, tools like Deep Learning (DL) and Machine Learning (ML) have enabled early detection of desirable traits in a large population of breeding material and mitigate plant stresses. In this review, advanced applications of ML and DL in plant biotic and abiotic stress management have been summarized.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Effects of Perfluorinated Alkyl Substances (PFAS) on Amphibian Body and Liver Conditions: Is Lipid Metabolism Being Perturbed throughout Metamorphosis?

Per- and polyfluoroalkyl substances (PFAS) may interact with peroxisome proliferator activated receptors (PPARs) and alter lipid homeostasis. Using Xenopus laevis, we investigated the effect of PFAS on (a) lipid homeostasis and whether this correlated to changes in body and hepatic condition; (b) the expression of hepatic genes regulated by PPAR; and (c) the hepatic lipidome. We chronically exposed tadpoles to 0.5 µg/L of either PFOS, PFHxS, PFOA, PFHxA, a binary mixture of PFOS and PFHxS (0.5 µg/L of each), or a control, from NF stage 52 through metamorphic climax. Growth, development, and survival were not affected, but we detected a sex-specific decrease in body condition at NF 66 (6.8%) and in hepatic condition (16.6%) across metamorphic climax for male tadpoles exposed to PFOS. We observed weak evidence for the transient downregulation of apolipoprotein-V (apoa5) at NF 62 in tadpoles exposed to PFHxA. Acyl-CoA oxidase 1 (acox1) was downregulated only in males exposed to PFHxS (Ln(Fold Change) = −0.54). We detected PFAS-specific downregulation of structural glycerophospholipids, while semi-quantitative profiling detected the upregulation in numerous glycerophospholipids, sphingomyelins, and diglycerides. Overall, our findings indicate that PFAS can induce sex-specific effects that change across larval development and metamorphosis. We demonstrate that PFAS alter lipid metabolism at environmentally relevant concentrations through divergent mechanisms that may not be related to PPARs, with an absence of effects on body condition, demonstrating the need for more molecular studies to elucidate mechanisms of PFAS-induced lipid dysregulation in amphibians and in other taxa.

Bushong, Anna (ORCID:0000000249630578)↗
DOE OSTI2020

12-gene prognostic signature for breast cancer survival

A biomarker panel of 12 genes based on expression levels. Methods for calculating prognostic scores and patient ranking based on their score and divided into two equal sized cohorts. Kaplan-Meier analysis and a log-rank test were used to determine differences in survival.

Snijders, Antoine M.↗
DOE OSTI2023

ABF DFO with Technology Holding, Inc.

This Agile BioFoundry Directed Funding Opportunity project with Technology Holding and partners focuses on the development of both a strain of Pseudomonas putida KT2440 and a corresponding bioprocess to convert cellulosic sugars to beta-ketoadipic acid, which can be used in performance nylons and polyesters. Our approach follows the Design-Build-Test-Learn cycle wherein we have transferred learnings from muconic acid production in P. putida to develop a glucose and xylose-utilizing beta-ketoadipic acid production strain. This strain achieves 65 g/L of beta-ketoadipic acid at 0.7 g/L/hr and a C-mol yield of 0.40. We are currently on-boarding arabinose utilization as well. To identify non-intuitive strain modifications as well, we are deploying a beta-ketoadipic acid biosensor and building randomly barcoded transposon insertion sequencing (RB-TnSeq) libraries and gene over-expression libraries in beta-ketoadipic acid production strains. Moreover, we are using global metabolomics and other systems biology tools to identify off-target pathways. Lastly, we are scaling up beta-ketoadipic acid production to kg-scale production for Technology Holding to evaluate in performance polymers with their partners.

beta-ketoadipic acid↗
DOE OSTI2024

Developing multi-gene CRISPRa/i programs to accelerate DBTL cycles in ABF hosts engineered for chemical production

This project developed and implemented a modular CRISPR activation and interference (CRISPRa/i) platform to accelerate strain optimization and pathway development for industrially relevant microbial hosts. By integrating multiplexed transcriptional perturbation tools with data-driven Design–Build–Test–Learn (DBTL) workflows, the team achieved reductions in cycle time and enhanced production of industrial aromatics, particularly 4-aminocinnamic acid (4-ACA), in Pseudomonas putida. Key accomplishments included: ● Development of a robust, tunable CRISPRa/i system in P. putida that enabled efficient multi-target gene regulation via guide RNA (gRNA) programs ● Completion of two full DBTL cycles, guided by machine learning (ML) models trained on transcriptomic and performance data, reducing engineering time by over 30% ● Optimization of multi-gene regulatory programs to balance expression of host and pathway modules, improve 4-ACA titers, and resolve metabolic bottlenecks ● Demonstration of system portability through a limited proof-of-concept extension in Acinetobacter baylyi, underscoring the generalizability of the approach ● Evaluation of strain performance on lignocellulosic biomass-derived substrates, demonstrating the feasibility of converting renewable carbon into aromatic building blocks These results illustrate the feasibility of applying ML-guided CRISPRa/i perturbation strategies to accelerate strain development in complex microbial systems. The resulting tools and datasets contribute to DOE objectives by improving platform predictability, reducing development costs, and enabling broader access to sustainable, economically viable bioproduction technologies.

09 BIOMASS FUELS↗
DOE OSTI2026

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

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

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The landscape of Chlamydomonas histone H3 lysine 4 methylation reveals both constant features and dynamic changes during the diurnal cycle

Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. Here, we interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G 1 phase, Zeitgeber Time 1 (ZT1), when cells initiate biomass accumulation, S/M phase (ZT13) when cells are replicating DNA and undergoing mitosis, and late G 0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at transcription start sites of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at some point during the life cycle but not necessarily by continuous active transcription, as exemplified by early zygotic genes, which may remain transcriptionally dormant for thousands of generations between sexual cycles. The exceptions to this rule were around 120 loci, some of which encode non-poly-adenylated transcripts, such as small nuclear RNAs and replication-dependent histones that had H3K4me3 peaks only when they were being transcribed. Mono-methylated H3K4 was the default state for the vast majority of histones that were bound outside of transcription start sites and terminator regions of genes. A small fraction of the genome that was depleted of any H3 lysine 4 methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Besides marking protein coding genes, H3K4me3 ChIP-seq data served also as a annotation tool for validation of hundreds of long non-coding RNA genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative proteomics of a versatile, marine, iron-oxidizing chemolithoautotroph

This study conducted a comparative proteomic analysis to identify potential genetic markers for the biological function of chemolithoautotrophic iron oxidation in the marine bacterium Ghiorsea bivora. To date, this is the only characterized species in the class Zetaproteobacteria that is not an obligate iron-oxidizer, providing a unique opportunity to investigate differential protein expression to identify key genes involved in iron-oxidation at circumneutral pH. Over 1000 proteins were identified under both iron- and hydrogen-oxidizing conditions, with differentially expressed proteins found in both treatments. Notably, a gene cluster upregulated during iron oxidation was identified. This cluster contains genes encoding for cytochromes that share sequence similarity with the known iron-oxidase, Cyc2. Interestingly, these cytochromes, conserved in both Bacteria and Archaea, do not exhibit the typical β-barrel structure of Cyc2. This cluster potentially encodes a biological nanowire-like transmembrane complex containing multiple redox proteins spanning the inner membrane, periplasm, outer membrane, and extracellular space. The upregulation of key genes associated with this complex during iron-oxidizing conditions was confirmed by quantitative reverse transcription-PCR. These findings were further supported by electromicrobiological methods, which demonstrated negative current production by G. bivora in a three-electrode system poised at a cathodic potential. This research provides significant insights into the biological function of chemolithoautotrophic iron oxidation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Transient knockdown of Anopheles stephensi LRIM1 using RNAi increases Plasmodium falciparum sporozoite salivary gland infections

Abstract Background Plasmodium falciparum (Pf) sporozoites (PfSPZ) can be administered as a highly protective vaccine conferring the highest protection seen to date. Sanaria® PfSPZ vaccines are produced using aseptically reared Anopheles stephensi mosquitoes. The bionomics of sporogonic development of P. falciparum in A. stephensi to fully mature salivary gland PfSPZ is thought to be modulated by several components of the mosquito innate immune system. In order to increase salivary gland PfSPZ infections in A. stephensi and thereby increase vaccine production efficiency, a gene knock down approach was used to investigate the activity of the immune deficiency (IMD) signaling pathway downstream effector leucine-rich repeat immune molecule 1 ( LRIM1 ), an antagonist to Plasmodium development. Methods Expression of LRIM1 in A. stephensi was reduced following injection of double stranded (ds) RNA into mosquitoes. By combining the Gal4/UAS bipartite system with in vivo expression of short hairpin (sh) RNA coding for LRIM1 reduced expression of LRIM1 was targeted in the midgut, fat body, and salivary glands. RT-qPCR was used to demonstrate fold-changes in gene expression in three transgenic crosses and the effects on P. falciparum infections determined in mosquitoes showing the greatest reduction in LRIM1 expression. Results LRIM1 expression could be reduced, but not completely silenced, by expression of LRIM1 dsRNA. Infections of P. falciparum oocysts and PfSPZ were consistently and significantly higher in transgenic mosquitoes than wild type controls, with increases in PfSPZ ranging from 2.5- to tenfold. Conclusions Plasmodium falciparum infections in A. stephensi can be increased following reduced expression of LRIM1. These data provide the springboard for more precise knockout of LRIM1 for the eventual incorporation of immune-compromised A. stephensi into manufacturing of Sanaria’s PfSPZ products.

Billingsley, Peter F. (ORCID:0000000311424813)↗
DOE OSTI2022

Temperature modulation of CAMTA3 gene induction activity is mediated through the DNA binding domain

The calmodulin-binding transcription activator (CAMTA) proteins of Arabidopsis thaliana play a major role in cold acclimation, contributing to the rapid induction of the C-REPEAT BINDING FACTOR (CBF) genes and other genes that impart freezing tolerance in plants exposed to cold temperature (4°C). The goal of this study was to better understand how the gene induction activity of CAMTA3 is modulated by temperature. Our results indicate that a severely truncated version of CAMTA3, CAMTA3 334 , which includes the N-terminal CG-1 DNA binding domain and a newly identified transcriptional activation domain (TAD), was able to rapidly induce the expression of CBF2 and two newly identified target genes, EXPANSIN-LIKE A1 (EXPL1) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), in response to cold temperature. Additionally, CAMTA3 334 was able to restore freezing tolerance when expressed in a camta23 double mutant. The ability of CAMTA3 and CAMTA3 334 to induce target genes at cold temperature did not involve increased levels of these proteins or increased binding of these proteins to target gene promoters in cold-treated plants. Rather, domain-swapping experiments indicated that the CAMTA3 CG-1 domain conferred temperature dependence to the ability of the CAMTA3 TAD to induce gene expression. The CG-1 domain also enabled the TAD to induce the expression of target genes at a moderate temperature (22°C) in response to cycloheximide treatment, consistent with the TAD activity not being intrinsically temperature dependent. We propose a working model in which the temperature modulation of CAMTA3 gene induction activity occurs independently from the C-terminal calmodulin-binding domains that previously have been proposed to activate CAMTA3 transcriptional activity in response to cold temperature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Omics-Lethal Human Viruses, Interferon-Stimulated Response Experiment IFNaHUH001

The purpose of this experiment was to evaluate the human host cellular response to treatment with and without interferon alpha/beta (IFNα/β) treatment. Sample time course data was obtained from human hepatoma carcinoma cells (HUH-7) expressing transcriptional activation protein VP30, stably transfected with the Ebola VP30 gene for enabling replication of VP30-deficient Ebola virus for transcriptome expression analysis. Secondary host-associated viral dataset downloads contain one or more statistically processed (normalization data transformation) quantitative dataset collections resulting in qualitative expression analyses of primary host-pathogen experimental study designs. Transcriptomics dataset download have a direct relationship to a primary sample submission corresponding to a specific interferon response to viral infection.

59 BASIC BIOLOGICAL SCIENCES↗