Engineering topics

Engbrecht, Kristin M.

Publications and source records attributed to Engbrecht, Kristin M..

7 records

DOE OSTI2024

Chemical Imaging for In Situ Detection and Discrimination of Aquatic Toxins Targeting Voltage Gated Sodium Channels

Biologically derived neurotoxins from cyanobacteria and algae impact environmental resources in addition to being considered a potential biological threat to human and animal health. Activity based assays are essential to detecting and responding to toxic neurotoxin events either naturally occurring or deliberate. Two toxins of interest include saxitoxin and brevetoxin. These toxins bind to and alter the function of voltage-gated sodium channels (NaV channels) which are essential for generating cell membrane action potential. We report the development and refinement of a System for Analysis at Liquid Vacuum Interface (SALVI) to assess the functional activity of saxitoxin and brevetoxin. This approach utilizes a vacuum-compatible microfluidic reactor that permits analysis at the liquid vacuum interface of human derived cells with a neurotoxin of interest in a biologically relevant environment.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Parallel measurement of transcriptomes and proteomes from same single cells using nanodroplet splitting

Single-cell multiomics provides comprehensive insights into gene regulatory networks, cellular diversity, and temporal dynamics. While tools for co-profiling single-cell genomes, transcriptomes, and epigenomes are available, accessing proteomes in parallel is more challenging. We developed nanoSPLITS (nanodroplet SPlitting for Linked-multimodal Investigations of Trace Samples), an integrated platform that enables global profiling of the transcriptome and proteome from same single cells using RNA sequencing and mass spectrometry-based proteomics, respectively. nanoSPLITS can precisely quantify over 5000 genes, 2000 proteins, and 140 phosphopeptides per single cell and identify candidate cell markers from these modalities. By exploring Cdk1-mediated cell cycle arrest, we demonstrate how nanoSPLITS single-cell multiomics can provide comprehensive cellular characterization with insights into covarying protein/gene clusters, unique phosphorylation events, and mitotic pathways.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Insights from a workplace SARS-CoV-2 specimen collection program, with genomes placed into global sequence phylogeny

In 2020, the Department of Energy established the National Virtual Biotechnology Laboratory (NVBL) to address key challenges associated with COVID-19. As part of that effort, Pacific Northwest National Laboratory (PNNL) established a capability to collect and analyze specimens from employees who self-reported symptoms consistent with the disease. During the spring and fall of 2021, 688 specimens were screened for SARS-CoV-2, with 64 (9.3%) testing positive using reverse-transcriptase quantitative PCR (RT-qPCR). Of these, 36 samples were released for research. All 36 positive samples released for research were sequenced and genotyped. Here, the relationship between patient age and viral load as measured by Ct values was measured and determined to be only weakly significant. Consensus sequences for each sample were placed into a global phylogeny and transmission dynamics were investigated, revealing that the closest relative for many samples was from outside of Washington state, indicating mixing of viral pools within geographic regions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Defining Lipidomic Responses to Coronavirus Infection

Highly pathogenic human coronavirus infection can cause a severe atypical, rapid onset pneumonia with a mortality rate of up to 10% for severe acute respiratory syndrome coronavirus 1 (SARS-CoV 1), 35% for Middle East respiratory syndrome coronavirus (MERS-CoV), or 1% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2 causative agent of COVID 19). While medical countermeasures successfully controlled the worldwide SARS-CoV epidemic, the MERS-CoV epidemic is still ongoing and continues to be a concern for travelers in the Middle East and the multi-year SARS-CoV 2 pandemic underscore the importance of defining biomarkers that are diagnostic and/or predictive of severe disease outcomes for respiratory viruses. Systems biology approaches provide global snapshots of infection induced changes in host cells/tissues and provide extremely rich datasets for understanding host pathogen interactions. Metabolites, especially lipids, are critical for viral replication but less is understood about infection induced changes to lipids due to limits in lipid species detection and identification. To characterize how individual lipid species and proteins with lipid associated functions contribute to highly pathogenic human coronavirus replication and disease severity, existing datasets were probed to determine cell type specific lipid responses to MERS-CoV infection and verification studies were performed to determine if modification of the host lipid signature (by inhibiting enzymatic functions that produce specific lipid species) can perturb CoV replication in human lung cells. MERS-CoV infects human lung epithelial, endothelial and fibroblast cells. All three cell types were infected with MERS-CoV and samples collected to analyze lipids, proteins, metabolites, and transcripts from 12 to 48 hours post infection. Time matched mock-infected cells were collected in parallel for each cell type. Following sample and statistical analysis, functional enrichment and bioinformatic analysis was performed to identify differentially expressed lipids and proteins. Two lipid species were found to be significantly upregulated following MERS-CoV infection, ceramides, and triacylglycerol both of which are indicative of cells undergoing apoptotic cell death. In contrast, sphingomyelins (lipid molecules that can serve as a precursor for one pathway for ceramide synthesis) had significantly decreased expression in MERS-CoV infected cells. An inhibitor of acid sphingomyelinase (that converts sphingomyelin to ceramides and phosphorylcholine) reduced MERS-CoV replication suggesting that production of ceramides is key for successful viral replication and transmission. Acyl-CoA-synthetase 3 (ACSL3), the only protein whose function is lipid associated and had increased differential expression in our dataset, regulates the synthesis of triacylglycerol (increased expression). Inhibitors that directly block ACSL3 (Triacsin C) but not steps later in the triacylglycerol synthesis pathway (Etomoxir) inhibit MERS-CoV replication suggesting that triacylglycerol production and/or ACSL3 activity is also key for viral replication. ACSL3 expression is also upregulated in lung cancer cells and is predicted to promote continued cell viability which would also enhance viral replication. The differentially expressed lipid and lipid-associated protein species identified in our studies suggest that MERS-CoV infection is activating cellular death pathways to limit the number of cells that become infected but also stimulating the production of lipid-associated enzymes that can prolong host cell viability and the amount of time progeny virions can be produced and released. As the inhibitors that worked against MERS-CoV infection were also efficacious against SARS-CoV 2 infection, countermeasures that target these host pathways may provide novel ways to block highly pathogenic human coronavirus infection and/or prevent severe disease outcomes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Biological Response Models: Predicting Injury and Mortality of Fish During Downstream Passage through Hydropower Facilities

This report describes the U.S. Department of Energy’s HydroPASSAGE project efforts to develop and collect biological response models for integration into the Biological Performance Assessment (BioPA) toolset and the Hydropower Biological Evaluation Toolset (HBET). These models help understand how fish are likely to respond during dam passage when exposed to hydraulic and physical stressors associated with turbines and other hydropower structures. When fish pass through hydropower facilities, they may encounter several stressors, of which the three most common are collision, rapid decompression, and fluid shear. Specialized equipment has been used to develop 99 biological response models for exposure to blade strike, rapid decompression, or fluid shear. The models were gathered from the literature or developed as part of this effort and include models for 31 different species of fish that have various predicted endpoints (i.e., injury or mortality). Among these models, considerable variation in susceptibility to the stressors has been observed from one species to another, and a species’ susceptibility to one stressor does not necessarily indicate similar susceptibility to another. Although several species have been examined, it is still unclear how many other species, which may have different morphological traits, may respond to these stressors, so further examination of the different species is needed. These models can and have been applied in several different cases, often using the BioPA toolset and HBET, to better understand the potential for injury and mortality that may occur during fish passage at hydropower facilities, including specific applications, such as turbine replacement, the installation of new turbines, or changing operations of currently installed turbines. As hydropower is continually developed to meet the electricity needs of society, tools such as HBET and BioPA, used with the integrated biological response models, will aid in the development of technologies and strategies that avoid, minimize, mitigate, or manage environmental effects.

13 HYDRO ENERGY↗

DOE OSTI2020

Distinct Preflowering Drought Tolerance Strategies of Sorghum bicolor Genotype RTx430 Revealed by Subcellular Protein Profiling

Drought is the largest stress affecting agricultural crops, resulting in substantial reductions in yield. Plant adaptation to water stress is a complex trait involving changes in hormone signaling, physiology, and morphology. Sorghum (Sorghum bicolor (L.) Moench) is a C4 cereal grass; it is an agricultural staple, and it is particularly drought-tolerant. To better understand drought adaptation strategies, we compared the cytosolic- and organelle-enriched protein profiles of leaves from two Sorghum bicolor genotypes, RTx430 and BTx642, with differing preflowering drought tolerances after 8 weeks of growth under water limitation in the field. In agreement with previous findings, we observed significant drought-induced changes in the abundance of multiple heat shock proteins and dehydrins in both genotypes. Interestingly, our data suggest a larger genotype-specific drought response in protein profiles of organelles, while cytosolic responses are largely similar between genotypes. Organelle-enriched proteins whose abundance significantly changed exclusively in the preflowering drought-tolerant genotype RTx430 upon drought stress suggest multiple mechanisms of drought tolerance. These include an RTx430-specific change in proteins associated with ABA metabolism and signal transduction, Rubisco activation, reactive oxygen species scavenging, flowering time regulation, and epicuticular wax production. We discuss the current understanding of these processes in relation to drought tolerance and their potential implications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Hydrologic Evaluation of the Ringold Upper Mud Aquifer in the 100-H Area of the Hanford Site

Aquifer tests were recently performed in the Hanford Site 100-H Area to provide a hydrologic evaluation of the Ringold Formation member of Wooded Island – upper mud unit (RUM) aquifer to reduce uncertainty regarding its lateral continuity and characterize aquifer properties within the high-concentration portion of the RUM Cr(VI) plume. A constant-rate pumping test was performed in well 199-H3-22 in October 2019. Additionally, the hydraulic responses from pump and treat (P&T) shutdown events occurring in June, July, and October 2019 were analyzed for aquifer hydraulic properties.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗