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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 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

Global LC/MS Metabolomics Profiling of Calcium Stressed and Immunosuppressant Drug Treated Saccharomyces cerevisiae

Previous studies have shown that calcium stressed Saccharomyces cerevisiae , challenged with immunosuppressant drugs FK506 and Cyclosporin A, responds with comprehensive gene expression changes and attenuation of the generalized calcium stress response. Here, we describe a global metabolomics workflow for investigating the utility of tracking corresponding phenotypic changes. This was achieved by efficiently analyzing relative abundance differences between intracellular metabolite pools from wild-type and calcium stressed cultures, with and without prior immunosuppressant drugs exposure. We used pathway database content from WikiPathways and YeastCyc to facilitate the projection of our metabolomics profiling results onto biological pathways. A key challenge was to increase the coverage of the detected metabolites. This was achieved by applying both reverse phase (RP) and aqueous normal phase (ANP) chromatographic separations, as well as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources for detection in both ion polarities. Unsupervised principle component analysis (PCA) and ANOVA results revealed differentiation between wild-type controls, calcium stressed and immunosuppressant/calcium challenged cells. Untargeted data mining resulted in 247 differentially expressed, annotated metabolites, across at least one pair of conditions. A separate, targeted data mining strategy identified 187 differential, annotated metabolites. All annotated metabolites were subsequently mapped onto curated pathways from YeastCyc and WikiPathways for interactive pathway analysis and visualization. Dozens of pathways showed differential responses to stress conditions based on one or more matches to the list of annotated metabolites or to metabolites that had been identified further by MS/MS. The purine salvage, pantothenate and sulfur amino acid pathways were flagged as being enriched, which is consistent with previously published literature for transcriptomics analysis. Thus, broad discovery-based data mining combined with targeted pathway projections can be an important asset for rapidly distilling, testing and evaluating a large amount of information for further investigation.

calcineurin↗
DOE OSTI2016

Quorum Quenching of Nitrobacter winogradskyi Suggests that Quorum Sensing Regulates Fluxes of Nitrogen Oxide(s) during Nitrification

ABSTRACT Quorum sensing (QS) is a widespread process in bacteria used to coordinate gene expression with cell density, diffusion dynamics, and spatial distribution through the production of diffusible chemical signals. To date, most studies on QS have focused on model bacteria that are amenable to genetic manipulation and capable of high growth rates, but many environmentally important bacteria have been overlooked. For example, representatives of proteobacteria that participate in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, produce QS signals called acyl-homoserine lactones (AHLs). Nitrification emits nitrogen oxide gases (NO, NO 2 , and N 2 O), which are potentially hazardous compounds that contribute to global warming. Despite considerable interest in nitrification, the purpose of QS in the physiology/ecology of nitrifying bacteria is poorly understood. Through a quorum quenching approach, we investigated the role of QS in a well-studied AHL-producing nitrite oxidizer, Nitrobacter winogradskyi . We added a recombinant AiiA lactonase to N. winogradskyi cultures to degrade AHLs to prevent their accumulation and to induce a QS-negative phenotype and then used mRNA sequencing (mRNA-Seq) to identify putative QS-controlled genes. Our transcriptome analysis showed that expression of nirK and nirK cluster genes ( ncgABC ) increased up to 19.9-fold under QS-proficient conditions (minus active lactonase). These data led to us to query if QS influenced nitrogen oxide gas fluxes in N. winogradskyi . Production and consumption of NO x increased and production of N 2 O decreased under QS-proficient conditions. Quorum quenching transcriptome approaches have broad potential to identify QS-controlled genes and phenotypes in organisms that are not genetically tractable. IMPORTANCE Bacterial cell-cell signaling, or quorum sensing (QS), is a method of bacterial communication and gene regulation that is well studied in bacteria. However, little is known about the purpose of QS in many environmentally important bacteria. Here, we demonstrate quorum quenching coupled with mRNA-Seq to identify QS-controlled genes and phenotypes in Nitrobacter winogradskyi , a nitrite-oxidizing bacterium. Nitrite oxidizers play an important role in the nitrogen cycle though their participation in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite. Our quorum quenching approach revealed that QS influences production and consumption of environmentally important nitrogen oxide gases (NO, NO 2 , and N 2 O) in N. winogradskyi . This study demonstrated a novel technique for studying QS in difficult-to-work-with microorganisms and showed that nitrite oxidizers might also contribute to nitrification-dependent production of nitrogen oxide gases that contribute to global warming.

Mellbye, Brett L.↗
DOE OSTI2024

Exploring the Structural, Biochemical, and Functional Diversity of Glycoside Hydrolase Family 12 from Penicillium subrubescens

Glycoside hydrolases (GHs) play an essential role in plant biomass degradation and modification for the sustainable production of biochemicals. The filamentous Ascomycete fungus Penicillium subrubescens contains a higher number of GH12 candidates compared to related species. Therefore, we aimed to compare P. subrubescens GH12s for their ability and substrate specificity for plant cell wall polysaccharide degradation and species’ potential as a source of novel enzymes for plant biomass valorization. Our re-evaluated phylogenetic analysis of fungal GH12 members showed that the P. subrubescens GH12s were located in different (new) clades. Biochemical characterization marked PsEglA as an endoglucanase and four other P. subrubescens GH12s (i.e., PsXegA–D) as xyloglucanases. Interestingly, structural features of PsXegD and PsXegE were more comparable to those of Basidiomycete GH12 xyloglucanases with a unique open substrate-binding cleft. PsUegA displayed dual xyloglucanase and endoglucanase activity and also showed distinct structural features. Comparative transcriptome analysis supported the functional diversity of P. subrubescens GH12s in plant biomass degradation. The gene encoding PsUegA was expressed under diverse conditions, suggesting a scouting role for this enzyme.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Feed status and skin injury modulate immunopathology, global gene expression, and survival in channel catfish during virulent Aeromonas hydrophila infection

Introduction VirulentAeromonas hydrophilais a major pathogen in channel catfish (Ictalurus punctatus), that causes motileAeromonassepticemia and significant economic losses. We investigated the effect of feeding status and skin integrity on the host immune response, disease survival, and gastrointestinal pathology following a vAh challenge. Methods Using a bath immersion model, channel catfish were divided into four treatment groups: fin clipped and fed (FCF), fin clipped but not fed (FCN), not fin clipped but fed (NCF), and not fin clipped nor fed (NCN) alongside non-challenged control groups The FCF and NCF groups were fed 2 h prior to the challenge, but the FCN and NCN groups were not. Survival analysis, histopathological assessment, and RNA sequencing were conducted across groups at different time intervals throughout the vAh challenge. Results Survival rates were lowest in the FCF and FCN groups (30% and 23% survival, respectively), suggesting that both feeding and skin damage contributed to disease severity. Histopathological analyses revealed more severe intestinal and gastric lesions in fed groups, characterized by epithelial necrosis, hemorrhage, and edema. Transcriptomic analysis among the groups identified significant differentially expressed genes associated with inflammation, apoptosis, and metabolic stress, with notable upregulation of interleukin 1-beta (il-1β), and complement C3 (c3). Gene ontology enrichment highlighted distinct immune activation patterns between fed and unfed groups, with enhanced pathogen recognition and pro-inflammatory responses in unfed fish. Discussion These findings suggest feeding prior to infection may exacerbate disease pathology, potentially by creating a physiological state conducive to facilitate pathogen proliferation and dampened early immune responses, whereas short-term fasting appears to promote early immune activation. This study provides novel insights into the complex interplay between feed status, physical injury, and immune response to vAh infection.

Immunology↗
DOE OSTI2016

Better Efficacy of Synchrotron Spatially Microfractionated Radiation Therapy Than Uniform Radiation Therapy on Glioma

Purpose: Synchrotron microbeam radiation therapy (MRT) is based on the spatial fractionation of the incident, highly focused synchrotron beam into arrays of parallel microbeams, typically a few tens of microns wide and depositing several hundred grays. This irradiation modality was shown to have a high therapeutic impact on tumors, especially in intracranial locations. However, mechanisms responsible for such a property are not fully understood. Methods and Materials: Thanks to recent progress in dosimetry, we compared the effect of MRT and synchrotron broad beam (BB) radiation therapy delivered at comparable doses (equivalent to MRT valley dose) on tumor growth control and on classical radiobiological functions by histologic evaluation and/or transcriptomic analysis. Results: MRT significantly improved survival of rats bearing 9L intracranial glioma compared with BB radiation therapy delivered at a comparable dose (P<.001); the efficacy of MRT and BB radiation therapy was similar when the MRT dose was half that of BB. The greater efficacy of MRT was not correlated with a difference in cell proliferation (Mki67 and proliferating cell nuclear antigen) or in transcriptomic stimulation of angiogenesis (vascular endothelial growth factor A or tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2) but was correlated with a higher cell death rate (factor for apoptosis signals) and higher recruitment of macrophages (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and CD68 transcripts) a few days after MRT. Conclusions: These results show the superiority of MRT over BB radiation therapy when applied at comparable doses, suggesting that spatial fractionation is responsible for a specific and particularly efficient tissue response. The higher induction of cell death and immune cell activation in brain tumors treated by MRT may be involved in such responses.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2024

Identification and functional analysis of strigolactone pathway genes regulating tillering traits in sugarcane

Abstract Saccharum officinarum and Saccharum spontaneum are two fundamental species of modern sugarcane cultivars, exhibiting divergent tillering patterns crucial for sugarcane architecture and yield. Strigolactones (SLs), a class of plant hormones, are considered to play a central role in shaping plant form and regulating tillering. Our study highlights the distinct tillering patterns observed between S. officinarum and S. spontaneum and implicates significant differences in SL levels in root exudates between the two species. Treatment with rac-GR24 (an artificial SL analog) suppressed tillering in S. spontaneum. Based on transcriptome analysis, we focused on two genes, TRANSCRIPTION ELONGATION FACTOR 1 (TEF1) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1), which show higher expression in S. spontaneum or S. officinarum, respectively. While the overexpression of SoCCA1 did not lead to significant phenotypic differences, overexpression of SsTEF1 in rice stimulated tillering and inhibited plant height, demonstrating its role in tillering regulation. However, the overexpression of suggests that SoCCA1 may not be the key regulator of sugarcane tillering. Yeast one-hybrid assays identified four transcription factors (TFs) regulating SsTEF1 and four and five TFs regulating SsCCA1 and SoCCA1. This study provides a theoretical foundation for deciphering the molecular mechanisms underlying the different tillering behaviors between S. officinarum and S. spontaneum, providing valuable insights for the molecular-based design of sugarcane breeding strategies.

Qi, Yiying↗
DOE OSTI2026

Carbon source-induced changes in the transcriptional landscape of Novacetimonas hansenii

Novacetimonas hansenii is a model organism for bacterial cellulose (BC) production, a biopolymer with broad industrial applications. To better understand its carbon source-dependent metabolism, we conducted the first time-resolved transcriptomic analysis of N. hansenii ATCC 23769 grown on five carbon sources (glucose, fructose, mannitol, galactose, and sucrose) across five time points (24–120h). Glucose, fructose, and mannitol supported growth and efficient BC production, whereas galactose and sucrose resulted in poor yields, likely due to limited transport and enzymatic capacity. Solid-state NMR revealed that cellulose crystallinity and polymorphism were carbon source-dependent, with reduced bcsD expression correlating with lower crystallinity. Transcriptomic profiling showed distinct metabolic responses. Cultures containing glucose and mannitol activated core metabolic pathways, fructose induced a delayed adaptive shift, and galactose and sucrose triggered stress-related gene expression. These results highlight the metabolic plasticity of N. hansenii and offer insights for enhancing BC production through carbon source optomization.

Bacterial cellulose↗
DOE OSTI2017

β-Carboxysome bioinformatics: identification and evolution of new bacterial microcompartment protein gene classes and core locus constraints

Carboxysomes are bacterial microcompartments (BMCs) that enhance CO 2 fixation in all cyanobacteria. Structurally, carboxysome shell proteins are classified according to the type of oligomer formed: hexameric (BMC-H), trimeric (BMC-T) and pentameric (BMC-P) proteins. To understand the forces driving the evolution of the carboxysome shell, we conducted a bioinformatic study of genes encoding β-carboxysome shell proteins, taking advantage of the recent large increase in sequenced cyanobacterial genomes. In addition to the four well-established BMC-H (CcmK1-4) classes, our analysis reveals two new CcmK classes, which we name CcmK5 and CcmK6. CcmK5 is phylogenetically closest to CcmK3 and CcmK4, and the ccmK5 gene is found only in genomes lacking ccmK3 and ccmk4 genes. ccmK6 is found predominantly in heterocyst-forming cyanobacteria. The gene encoding the BMC-T homolog CcmO is associated with the main carboxysome locus (MCL) in only 60% of all species. We find five evolutionary origins of separation of ccmO from the MCL. Transcriptome analysis demonstrates that satellite ccmO genes, in contrast to MCL-associated ccmO genes, are never co-regulated with other MCL genes. The dispersal of carboxysome shell genes across the genome allows for distinct regulation of their expression, perhaps in response to changes in environmental conditions

regulation↗
DOE OSTI2018

Transcriptomic and phenotypic profiling in developing zebrafish exposed to thyroid hormone receptor agonists

There is a need to develop novel, high-throughput screening and prioritization methods to identify chemicals with adverse estrogen, androgen, and thyroid activity to protect human health and the environment and is of interest to the Endocrine Disruptor Screening Program. The current aim is to explore the utility of zebrafish as a testing paradigm to classify endocrine activity using phenotypically anchored transcriptome profiling. Transcriptome analysis was conducted on embryos exposed to 25 estrogen-, androgen-, or thyroid-active chemicals at a concentration that elicited adverse malformations or mortality at 120 hours post-fertilization in 80% of the animals exposed. Analysis of the top 1000 significant differentially expressed transcripts across all treatments identified a unique transcriptional and phenotypic profile for thyroid hormone receptor agonists, which can be used as a biomarker screen for potential thyroid hormone agonists.

endocrine disruptors↗
DOE OSTI2017

Multi-time series RNA-seq analysis of Enterobacter lignolyticus SCF1 during growth in lignin-amended medium

The production of lignocellulosic-derived biofuels is a highly promising source of alternative energy, but it has been constrained by the lack of a microbial platform capable to efficiently degrade this recalcitrant material and cope with by-products that can be toxic to cells. Species that naturally grow in environments where carbon is mainly available as lignin are promising for finding new ways of removing the lignin that protects cellulose for improved conversion of lignin to fuel precursors. Enterobacter lignolyticus SCF1 is a facultative anaerobic Gammaproteobacteria isolated from tropical rain forest soil collected in El Yunque forest, Puerto Rico under anoxic growth conditions with lignin as sole carbon source. Whole transcriptome analysis of SCF1 during E.lignolyticus SCF1 lignin degradation was conducted on cells grown in the presence (0.1%, w/w) and the absence of lignin, where samples were taken at three different times during growth, beginning of exponential phase, midexponential phase and beginning of stationary phase. Lignin-amended cultures achieved twice the cell biomass as unamended cultures over three days, and in this time degraded 60% of lignin. Transcripts in early exponential phase reflected this accelerated growth. A complement of laccases, aryl-alcohol dehydrogenases, and peroxidases were most up-regulated in lignin amended conditions in mid-exponential and early stationary phases compared to unamended growth. The association of hydrogen production by way of the formate hydrogenlyase complex with lignin degradation suggests a possible value added to lignin degradation in the future.

Orellana, Roberto (ORCID:0000000306333458)↗
DOE OSTI2015

TU-CD-BRB-10: 18F-FDG PET Image-Derived Tumor Features Highlight Altered Pathways Identified by Trancriptomic Analysis in Head and Neck Cancer

Purpose: Several quantitative features can be extracted from 18F-FDG PET images, such as standardized uptake values (SUVs), metabolic tumor volume (MTV), shape characterization (SC) or intra-tumor radiotracer heterogeneity quantification (HQ). Some of these features calculated from baseline 18F-FDG PET images have shown a prognostic and predictive clinical value. It has been hypothesized that these features highlight underlying tumor patho-physiological processes at smaller scales. The objective of this study was to investigate the ability of recovering alterations of signaling pathways from FDG PET image-derived features. Methods: 52 patients were prospectively recruited from two medical centers (Brest and Poitiers). All patients underwent an FDG PET scan for staging and biopsies of both healthy and primary tumor tissues. Biopsies went through a transcriptomic analysis performed in four spates on 4×44k chips (Agilent™). Primary tumors were delineated in the PET images using the Fuzzy Locally Adaptive Bayesian algorithm and characterized using 10 features including SUVs, SC and HQ. A module network algorithm followed by functional annotation was exploited in order to link PET features with signaling pathways alterations. Results: Several PET-derived features were found to discriminate differentially expressed genes between tumor and healthy tissue (fold-change >2, p<0.01) into 30 co-regulated groups (p<0.05). Functional annotations applied to these groups of genes highlighted associations with well-known pathways involved in cancer processes, such as cell proliferation and apoptosis, as well as with more specific ones such as unsaturated fatty acids. Conclusion: Quantitative features extracted from baseline 18F-FDG PET images usually exploited only for diagnosis and staging, were identified in this work as being related to specific altered pathways and may show promise as tools for personalizing treatment decisions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Association mapping, transcriptomics, and transient expression identify candidate genes mediating plant–pathogen interactions in a tree

Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To reflect how this process can be accelerated, we employed a genome-wide association mapping of ca. 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among crucial associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose–binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids are observed in NASH. ► Hepatic bile acid synthesis shifts toward the alternative pathway in NASH.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Study of the effect of the trancription factor SARO_RS14285 in aromatics degradation in Novosphingobium aromaticivorans

Novosphingobium aromaticivorans DSM12444 is a bacterium capable of catabolizing several lignin aromatics. A main degradation pathway for these compounds is the protocatechuate (PCA) meta-cleavage. Nevertheless, the transcriptional regulation of this pathway is still unknown. Immediately upstream of the genes encoding the enzymes for this pathway, the LysR-type transcription factor (LTTF) SARO_RS14285 was identified. To evaluate the functionality of this LTTF, a deletion mutant was constructed. A transcriptomic analysis of the mutant strain cultured in several aromatic compounds is included in this report. Overall design: RNA-seq profiling of the WT and the deletion mutant cultured in minimal medium with glucose and one of the following aromatics: protocatechuic acid (PCA), 4-coumaric acid (4-CA), vanillic acid (VA) or syringic acid (SA).

aromatics↗
DOE OSTI2012

Genome-Facilitated Analyses of Geomicrobial Processes

This project had the goal(s) of understanding the mechanism(s) of extracellular electron transport (EET) in the microbe Shewanella oneidensis MR-1, and a number of other strains and species in the genus Shewanella. The major accomplishments included sequencing, annotation, and analysis of more than 20 Shewanella genomes. The comparative genomics enabled the beginning of a systems biology approach to this genus. Another major contribution involved the study of gene regulation, primarily in the model organism, MR-1. As part of this work, we took advantage of special facilities at the DOE: e.g., the synchrotron radiation facility at ANL, where we successfully used this system for elemental characterization of single cells in different metabolic states (1). We began work with purified enzymes, and identification of partially purified enzymes, leading to initial characterization of several of the 42 c-type cytochromes from MR-1 (2). As the genome became annotated, we began experiments on transcriptome analysis under different conditions of growth, the first step towards systems biology (3,4). Conductive appendages of Shewanella, called bacterial nanowires were identified and characterized during this work (5, 11, 20,21). For the first time, it was possible to measure the electron transfer rate between single cells and a solid substrate (20), a rate that has been confirmed by several other laboratories. We also showed that MR-1 cells preferentially attach to cells at a given charge, and are not attracted, or even repelled by other charges. The interaction with the charged surfaces begins with a stimulation of motility (called electrokinesis), and eventually leads to attachment and growth. One of the things that genomics allows is the comparative analysis of the various Shewanella strains, which led to several important insights. First, while the genomes predicted that none of the strains looked like they should be able to degrade N-acetyl glucosamine (NAG), the monomer that makes up chitin, virtually all of the strains were in fact capable. This led to the discovery of a great many new genes involved with chitin and NAG metabolism (7). In a similar vein, a detailed study of the sugar utilization pathway revealed a major new insight into the regulation of sugar metabolism in this genus (19). Systems Biology and Comparative Genomics of the shewanellae: Several publications were put together describing the use of comparative genomics for analyses of the group Shewanella, and these were a logical culmination of our genomic-driven research (10,15,18). Eight graduate students received their Ph.D. degrees doing part of the work described here, and four postdoctoral fellows were supported. In addition, approximately 20 undergraduates took part in projects during the grant period.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2016

The Foot-and-Mouth Disease Carrier State Divergence in Cattle

The pathogenesis of persistent foot-and-mouth disease virus (FMDV) infection was investigated in 46 cattle that were either naive or had been vaccinated using a recombinant, adenovirus-vectored vaccine 2 weeks before challenge. The prevalence of FMDV persistence was similar in both groups (62% in vaccinated cattle, 67% in nonvaccinated cattle), despite vaccinated cattle having been protected from clinical disease. Analysis of antemortem infection dynamics demonstrated that the subclinical divergence between FMDV carriers and animals that cleared the infection had occurred by 10 days postinfection (dpi) in vaccinated cattle and by 21 dpi in nonvaccinated animals. The anatomic distribution of virus in subclinically infected, vaccinated cattle was restricted to the pharynx throughout both the early and the persistent phases of infection. In nonvaccinated cattle, systemically disseminated virus was cleared from peripheral sites by 10 dpi, while virus selectively persisted within the nasopharynx of a subset of animals. The quantities of viral RNA shed in oropharyngeal fluid during FMDV persistence were similar in vaccinated and nonvaccinated cattle. FMDV structural and nonstructural proteins were localized to follicle-associated epithelium of the dorsal soft palate and dorsal nasopharynx in persistently infected cattle. Host transcriptome analysis of tissue samples processed by laser capture microdissection indicated suppression of antiviral host factors (interferon regulatory factor 7, CXCL10 [gamma interferon-inducible protein 10], gamma interferon, and lambda interferon) in association with persistent FMDV. In contrast, during the transitional phase of infection, the level of expression of IFN- mRNA was higher in follicle-associated epithelium of animals that had cleared the infection. This work provides novel insights into the intricate mechanisms of FMDV persistence and contributes to further understanding of this critical aspect of FMDV pathogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Visolis Microbial Chemical Intermediate Library Screening (CRADA Final Report)

Visolis is developing a commercial process for the bioproduction of a chemical intermediate, towards the derivative production of a portfolio of bio-based chemicals with large application potential, from drop-in commodity chemicals, to innovative specialty chemical offerings, to materials for multiple end markets and industries, which will ultimately benefit consumers and the public. Visolis already has several variants of a microbe that produce the chemical intermediate at distinct levels (i.e. high, medium, and low). They are currently pursuing transcriptomics analysis for some of these strains, towards a better understanding of the biology behind what makes certain strains perform better than others. There exist genome-scale library approaches for the microbe that could be used to screen gene disruption, overexpression, or repression candidates for perturbations to the production of the chemical intermediate. These approaches often reveal opportunities for further production improvement that are not otherwise accessible using hypothesis-driven metabolic engineering approaches. However, Visolis, while it could generate or procure such genome-scale libraries, does not have the automated strain engineering workflows required to screen thousands of variants, obtaining a production phenotype and a genotype for each. The purpose of this collaboration is to use LBNL and SNL capabilities to enable Visolis to effectively screen thousands of genome-scale library strain variants for phenotype/genotype relationships that will complement Visolis’ transcriptomic investigations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion

The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.

59 BASIC BIOLOGICAL SCIENCES↗