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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Mammalian Chromosome Analysis and Sorting by Flow Cytometry

The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The application of flow cytometry for kelp meiospore isolation

The goal of this study was to evaluate flow-cytometric techniques for isolating individual kelp meiospores into 96-welled plates. Previously reported low success rates for sorting tens of thousands of meiospores at a time have been improved by technological developments, specifically, the low nozzle pressure of the commercially available, JSAN (Bay Bioscience Co. Ltd) instrument. We monitored growth and gametophyte development post-sorting for 10 months. Our data demonstrate that successful kelp meiospore isolations of up to 76% viability can be achieved with flow-cytometry. This method can save time as compared to traditional, manual isolations using pipettes and improves confidence that self-fertilized individuals will not contaminate specific crosses of resulting gametophytes. Our results highlight a new application for the flow cytometer to produce clonal kelp gametophytes with direct applications for germplasm and culture collection development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Predicting Anaerobic Membrane Bioreactor Performance Using Flow-Cytometry-Derived High and Low Nucleic Acid Content Cells

Having a tool to monitor the microbial abundances rapidly and to utilize the data to predict the reactor performance would facilitate the operation of an anaerobic membrane bioreactor (AnMBR). This study aims to achieve the aforementioned scenario by developing a linear regression model that incorporates a time-lagging mode. The model uses low nucleic acid (LNA) cell numbers and the ratio of high nucleic acid (HNA) to LNA cells as an input data set. First, the model was trained using data sets obtained from a 35 L pilot-scale AnMBR. The model was able to predict the chemical oxygen demand (COD) removal efficiency and methane production 3.5 days in advance. Subsequent validation of the model using flow cytometry (FCM)-derived data (at time t – 3.5 days) obtained from another biologically independent reactor did not exhibit any substantial difference between predicted and actual measurements of reactor performance at time t. Further cell sorting, 16S rRNA gene sequencing, and correlation analysis partly attributed this accurate prediction to HNA genera (e.g., Anaerovibrio and unclassified Bacteroidales) and LNA genera (e.g., Achromobacter, Ochrobactrum, and unclassified Anaerolineae). In summary, our findings suggest that HNA and LNA cell routine enumeration, along with the trained model, can derive a fast approach to predict the AnMBR performance.

42 ENGINEERING↗
DOE OSTI2020

Ultrasonic analyte concentration and application in flow cytometry

The present invention includes an apparatus and corresponding method for concentrating analytes within a fluid flowing through a tube using acoustic radiation pressure. The apparatus includes a function generator that outputs a radio frequency electrical signal to a transducer that transforms the radio frequency electric signal to an acoustic signal and couples the acoustic signal to the tube. The acoustic signal is converted within the tube to acoustic pressure that concentrates the analytes within the fluid.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2024

Evaluation of Inactivation Methods for Rift Valley Fever Virus in Mouse Microglia

Rift Valley fever phlebovirus (RVFV) is a highly pathogenic mosquito-borne virus with bioweapon potential due to its ability to be spread by aerosol transmission. Neurological symptoms are among the worst outcomes of infection, and understanding of pathogenesis mechanisms within the brain is limited. RVFV is classified as an overlap select agent by the CDC and USDA; therefore, experiments involving fully virulent strains of virus are tightly regulated. Here, we present two methods for inactivation of live virus within samples derived from mouse microglia cells using commercially available kits for the preparation of cells for flow cytometry and RNA extraction. Using the flow cytometry protocol, we demonstrate key differences in the response of primary murine microglia to infection with fully virulent versus attenuated RVFV.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

A flow cytometric assay to detect viability and persistence of Salmonella enterica subsp. enterica serotypes in nuclease-free water at 4 and 25°C

Salmonella spp. is one of the most isolated microorganisms reported to be responsible for human foodborne diseases and death. Water constitutes a major reservoir where the Salmonella spp. can persist and go undetected when present in low numbers. In this study, we assessed the viability of 12 serotypes of Salmonella enterica subsp. enterica for 160 days in nuclease-free water at 4 and 25°C using flow cytometry and Tryptic Soy Agar (TSA) plate counts. The results show that all 12 serotypes remain viable after 160 days in distilled water using flow cytometry, whereas traditional plate counts failed to detect ten serotypes incubated at 25°C. Moreover, the findings demonstrate that 4°C constitutes a more favorable environment where Salmonella can remain viable for prolonged periods without nutrients. Under such conditions, however, Salmonella exhibits a higher susceptibility to all tested antibiotics and benzalkonium chloride (BZK). The pre-enrichment with Universal Pre-enrichment Broth (UP) and 1/10 × Tryptic Soy broth (1/10 × TSB) resuscitated all tested serotypes on TSA plates, nevertheless cell size decreased after 160 days. Furthermore, phenotype microarray (PM) analysis of S. Inverness and S. Enteritidis combined with principal component analysis (PCA) revealed an inter-individual variability in serotypes with their phenotype characteristics, and the impact of long-term storage at 4 and 25°C for 160 days in nuclease-free water. This study provides an insight to Salmonella spp. long-term survivability at different temperatures and highlights the need for powerful tools to detect this microorganism to reduce the risk of disease transmission of foodborne pathogens via nuclease-free water.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Circulating Tumour Cell Numbers Correlate with Platelet Count and Circulating Lymphocyte Subsets in Men with Advanced Prostate Cancer: Data from the ExPeCT Clinical Trial (CTRIAL-IE 15-21)

Interactions between circulating tumour cells (CTCs) and platelets are thought to inhibit natural killer(NK)-cell-induced lysis. We attempted to correlate CTC numbers in men with advanced prostate cancer with platelet counts and circulating lymphocyte numbers. Sixty-one ExPeCT trial participants, divided into overweight/obese and normal weight groups on the basis of a BMI ≥ 25 or <25, were randomized to participate or not in a six-month exercise programme. Blood samples at randomization, and at three and six months, were subjected to ScreenCell filtration, circulating platelet counts were obtained, and flow cytometry was performed on a subset of samples (n = 29). CTC count positively correlated with absolute total lymphocyte count (r 2 = 0.1709, p = 0.0258) and NK-cell count (r 2 = 0.49, p < 0.0001). There was also a positive correlation between platelet count and CTC count (r 2 = 0.094, p = 0.0001). Correlation was also demonstrated within the overweight/obese group (n = 123, p < 0.0001), the non-exercise group (n = 79, p = 0.001) and blood draw samples lacking platelet cloaking (n = 128, p < 0.0001). By flow cytometry, blood samples from the exercise group (n = 15) had a higher proportion of CD3+ T-lymphocytes (p = 0.0003) and lower proportions of B-lymphocytes (p = 0.0264) and NK-cells (p = 0.015) than the non-exercise group (n = 14). These findings suggest that CTCs engage in complex interactions with the coagulation cascade and innate immune system during intravascular transit, and they present an attractive target for directed therapy at a vulnerable stage in metastasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Amazonian fog harbors viable microbes

Fog formation over tropical forests remains poorly characterized, despite its potential role in bioaerosol dispersion and ecosystem processes. Here, we analyzed fog samples collected at the Amazon Tall Tower Observatory using flow cytometry and culture-based techniques to characterize viable microbial communities. Microbial cell concentrations varied over an order of magnitude across 13 fog events, reaching up to 8 × 104 cells per ml of fog water. Flow cytometry consistently detected metabolically active cells, while culturing and mass spectrometry-based identification yielded eight viable bacterial species and seven fungal taxa. The bacteria Serratia marcescens, Ralstonia pickettii and Sphingomonas paucimobilis exhibited seasonal variations in prevalence. The fungal species identified were primarily mesophilic saprophytes and endophytes, commonly associated with soil and plant surfaces. Our findings indicate that fog harbors viable microbes, including Serratia marcescens and Ralstonia pickettii, which may imply a relevance of fog for microbial dispersal, colonization and nutrient cycling in the Amazon rainforest.

Godoi, Ricardo H. (ORCID:0000000247744870)↗
DOE OSTI2024

Nanobodies as potential tools for microbiological testing of live biotherapeutic products

Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Data for Protoplast Fusion as a Strategy to Increase Ploidy in Rhodotorula toruloides for Strain Development

Rhodotorula toruloides is a red oleaginous yeast with growing commercial interest because of its hardiness and exceptional lipid production capacity. Because it is a basidiomycete yeast with a complex life cycle, many of the classical breeding methods used with ascomycetes are unavailable for strain improvement. However, we have been able to construct polyploid yeast by fusing protoplasts of parents with the same mating type. Fusing of Y-6985 (A2) and Y-48190 (A2), which had been transformed with complementary antibiotic markers, led to the recovery of two diploids and one triploid. The stability of the fusion yeasts was tested by plating them on non-selective medium after several growth cycles under antibiotics and then testing five colonies per strain for nuclear DNA contents using flow cytometry and standard cell cycle analysis: the triploid and one diploid were stable. Fusants inherited their mitochondria from a single parent, which was demonstrated using restriction fragment length polymorphism (RFLP) of mitochondrial DNA. The phenotypic properties of the parents and fusants were compared in glucose fed-batch bioreactor studies and cellulosic sugar batch cultures. The final lipid titers for the fed-batch cultures were 24.9–39.7 g/L with Y-6985 and the diploid and triploid performing the best and worst, respectively. The fusants demonstrated intermediate hardiness for growth on hydrolysate prepared with dilute-acid pretreated switchgrass and were outperformed by Y-48190. Unlike one of the haploid parents, the fusants grew in 70% v/v concentrated hydrolysate. However, they did not grow as fast as the other haploid. In this study, a modernized protoplast fusion method is resurrected a useful tool for strain development in this yeast, which is complementary with other available methods.

FOS: Biological sciences↗
DOE OSTI2024

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Hill, John D.↗
DOE OSTI2020

Genetic identification of 91 poplar cultivars based on SSR markers

Accurate genetic identification and relationship analysis of poplar cultivars is necessary to establish commercial poplar plantations and select suitable breeding strategies. In this study, 91 poplar cultivars belonging to four sections (Aigeiros, Tacamahaca, Populus and Turanga) and inter/intra-sectional hybrids were genotyped using 18 polymorphic simple sequence repeat (SSR) markers. In total, 222 alleles were amplified with an average of 12.3 alleles per marker. The mean polymorphic information content and power of discrimination were 0.706 and 0.813, respectively. Five SSR markers (ORPM_103, ORPM_247, GCPM_1048, GCPM_1255 and LG_X_19) constituted a core fingerprint and were sufficient to identify all the tested cultivars. With some notable exceptions, cultivars of the same species generally clustered together in cluster (UPGMA) and ordination (PCO) analyses. Flow cytometry indicated that 11 poplar cultivars were triploid. Among these, seven had three alleles at some loci, suggesting that SSR markers could indicate the ploidy level to some extent. This study provides useful genetic information for the identification and protection of poplar cultivars in China and offers a guideline for the selection of poplar crossing parents based on ploidy level and genetic relationships.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Bioaerosol Emission Characteristics from Laboratory Burns

Combustion processes can aerosolize and transport particles both from nonbiological and biological origin, with the latter termed bioaerosol particle (BAP). Previous work has shown an increase in the level of BAP in smoke plumes. The mechanism of their emission, whether from combustion of biological material or coemission with dust and soil from fire-driven winds, has yet to be examined. Here, we carried out a series of controlled combustion experiments to understand the role of vegetation type and combustion conditions in the direct emission of BAP. The fuels we used included broadleaf, evergreen, and grass. We measured the emitted fluorescent BAP using a wideband integrated bioaerosol system (WIBS), and we measured the viability of filter-collected BAP using flow cytometry. Our measurements showed that the size and absolute concentration of the fluorescent BAP, and the viability of the BAP emitted during combustion, depend on the combustion conditions. In addition, the type of fuel impacted the type of emitted fluorescent BAP fraction relative to the total concentration of the emitted aerosol.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2019

Fluorescent Crimean-Congo hemorrhagic fever virus illuminates tissue tropism patterns and identifies early mononuclear phagocytic cell targets in Ifnar -/- mice

Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/β receptor knock-out (Ifnar -/- ) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in latestage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations.

59 BASIC BIOLOGICAL SCIENCES↗