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DOE OSTI2024

A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging

Abstract Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca 2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca 2+ concentration using fluorescence lifetime imaging microscopy (FLIM).

Biochemistry & Molecular Biology↗

DOE OSTI2023

High-throughput, dynamic, multi-dimensional: an expanding repertoire of plant respiration measurements

A recent burst of technological innovation and adaptation has greatly improved our ability to capture respiration rate data from plant sources. At the tissue level, several independent respiration measurement options are now available, each with distinct advantages and suitability, including high-throughput sampling capacity. These advancements facilitate the inclusion of respiration rate data into large-scale biological studies such as genetic screens, ecological surveys, crop breeding trials, and multi-omics molecular studies. As a result, our understanding of the correlations of respiration with other biological and biochemical measurements is rapidly increasing. Difficult questions persist concerning the interpretation and utilization of respiration data; concepts such as allocation of respiration to growth versus maintenance, the unnecessary or inefficient use of carbon and energy by respiration, and predictions of future respiration rates in response to environmental change are all insufficiently grounded in empirical data. However, we emphasize that new experimental designs involving novel combinations of respiration rate data with other measurements will flesh-out our current theories of respiration. Furthermore, dynamic recordings of respiration rate, which have long been used at the scale of mitochondria, are increasingly being used at larger scales of size and time to reflect processes of cellular signal transduction and physiological response to the environment. Herein, we also highlight how respiratory methods are being better adapted to different plant tissues including roots and seeds, which have been somewhat neglected historically.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Multi-Omics Reveals Temporal Scales of Carbon Metabolism in Synechococcus Elongatus PCC 7942 Under Light Disturbance

Central carbon metabolism in model cyanobacteria involves multiple pathways to adapt to energy-light limitations across diel cycles. However, the success in mechanistic modeling for phenotypic prediction of the protein regulators in the metabolic state depends on capturing the vast possibilities emerging from multiple regulatory pathways in complex biological processes. Here, we developed a physics-informed machine learning approach based on energy-landscape concepts to predict regulatory proteins responding to cyclic circadian and unforeseen light perturbations in cyanobacterial metabolic networks. Our approach provides interpretable de novo models for inferring gene expression dynamics from Synechococcus elongatus over diel cycles and using redox proteome analysis to distinguish immediate light-responsive elements from circadian-regulated processes in carbon metabolism pathways. We identified distinct temporal signatures with the analysis of the redox proteome: there was an immediate shift in cysteine redox states accompanied by a limited change in protein abundance under constant illumination and after 2 hours of darkness. This discovery indicates that the generation of reductants coordinates photoinduced electron transport with redox metabolic pathways in two discernable molecular mechanisms: fast redox-based protein modifications occur immediately after the light disturbance, followed by slow transcriptional regulations across networks. This temporal regulation reveals how metabolic networks integrate rapid light responses with programmed circadian rhythms to maintain cellular homeostasis under the light-energy limitations over the diel cycle.

Biomolecular & subcellular processes↗

DOE OSTI2025

Three-dimensional modeling of hyphal fusion, branching, and nutrient transport in filamentous fungi

Fungi exhibit behaviors distinct from other microbes. Filamentous fungi grow by extending complex networks of branched filaments collectively referred to as the mycelium. These networks can expand over large distances and traverse low-nutrient areas by translocating nutrients through the filament network. This spatial characteristic makes filamentous fungi crucial for soil ecosystems, supporting stable microbial communities and promoting plant growth. However, simulating these behaviors is complex. The elongated nature of fungal compartments results in different mechanical interactions compared to the commonly modeled spherical bacteria. These detailed hyphal mechanics require specialized consideration and are often excluded from conventional fungal simulation packages. Additionally, the extensive fungal networks in nature demand computationally intensive simulations, necessitating high-performance algorithms. Therefore, realistic fungi simulations require specialized software. Here, we introduce a fungal modeling expansion to the high-performance biological modelling and interface exchange (bmx) software suite. bmx leverages adaptive mesh refinement in AMReX for chemical diffusion and incorporates a full mechanical model for bacterial cells, accelerated by GPUs. By extending bmx to model filamentous particles, we demonstrate the formation of complex filament networks through interactions like hyphal branching and fusion (anastomosis). We show that the networks produced match real-world fungal structures through various metrics. This work supports computational studies of fungal growth dynamics and can be adapted to investigate the growth of other filamentous structures in biology or materials science. The expanded-BMX package is open-sourced and is available online.

Cell mechanics↗

NASA NTRS2011

Modeling Acute Health Effects of Astronauts from Exposure to Large Solar Particle Events

In space exploration outside the Earth s geomagnetic field, radiation exposure from solar particle events (SPE) presents a health concern for astronauts, that could impair their performance and result in possible failure of the mission. Acute risks are of special concern during extra-vehicular activities because of the rapid onset of SPE. However, most SPEs will not lead to acute risks but can lead to mission disruption if accurate projection methods are not available. Acute Radiation Sickness (ARS) is a group of clinical syndromes developing acutely (within several seconds to 3 days) after high dose whole-body or significant partial-body ionizing radiation exposures. The manifestation of these syndromes reflects the disturbance of physiological processes of various cellular groups damaged by radiation. Hematopoietic cells, skin, epithelium, intestine, and vascular endothelium are among the most sensitive tissues of human body to ionizing radiation. Most ARS symptoms are directly related to these tissues and other systems (nervous, endocrine, and cardiovascular, etc.) with coupled regulations. Here we report the progress in bio-mathematical models to describe the dose and time-dependent early human responses to ionizing radiation. The responses include lymphocyte depression, granulocyte modulation, fatigue and weakness syndrome, and upper gastrointestinal distress. The modest dose and dose-rates of SPEs are predicted to lead to large sparing of ARS, however detailed experimental data on a range of proton dose-rates for organ doses from 0.5 to 2 Gy is needed to validate the models. We also report on the ARRBOD code that integrates the BRYNTRN and SUMDOSE codes, which are used to estimate the SPE organ doses for astronauts under various space travel scenarios, with our models of ARS. The more recent effort is to provide easy web access to space radiation risk assessment using the ARRBOD code.

Hu, Shaowen↗

NASA NTRS2017

Role of Mitochondrial Oxidative Stress in Spaceflight-Induced Tissue Degeneration

Microgravity and ionizing radiation in the spaceflight environment poses multiple challenges to homeostasis and may contribute to cellular stress. Effects may include increased generation of reactive oxygen species (ROS), DNA damage and repair error, cell cycle arrest, cell senescence or death. Our central hypothesis is that prolonged exposure to the spaceflight environment leads to the excess production of ROS and oxidative damage, culminating in accelerated tissue degeneration. The main goal of this project is to determine the importance of cellular redox defense for physiological adaptations and tissue degeneration in the space environment.

transgenic mic↗

NASA NTRS2018

Aging and Spaceflight: Catalase Targeted to Mitochondria Alters Skeletal Structure and Responses to Musculoskeletal Disuse

Microgravity and ionizing radiation in the spaceflight environment pose multiple challenges to homeostasis and may contribute to cellular stress. Effects may include increased generation of reactive oxygen species (ROS), DNA damage and repair error, cell cycle arrest, cell senescence or death. Our central hypothesis is that prolonged exposure to the spaceflight environment leads to excess production of ROS and oxidative damage, culminating in accelerated tissue degeneration which resembles aging. The main goal of this project is to determine the importance of cellular redox defense for physiological adaptations and tissue degeneration in the space environment. To accomplish this, we will use both wildtype (WT) mice and a well-established, genetically-engineered animal model (mCAT mice) which displays extended lifespan (Schriner et al. 2005). The animal model selected to test these ideas is engineered to quench ROS in mitochondria by targeted over-expression of the human catalase gene to the mitochondrial matrix. We showed previously that mCAT mice express the catalase transgene in skeletal tissues, bone forming osteoblasts, and bone resorbing osteoclasts. In addition, mCAT mice also display increased catalase activity in bone. Our findings revealed that exposure of adult, male, C57Bl/6J mice to simulated spaceflight (hindlimb unloading and gamma radiation) led to an increase in markers of oxidative damage (malondialdehyde, 4-hydroxynonenol) in skeletal tissue of WT mice but not mCAT mice. To extend our hypothesis to other, spaceflight-relevant tissues, we are performing a ground-based study simulating 30 days of spaceflight by hindlimb unloading to determine potential protective effects of mitochondrial catalase activity on aging of multiple tissues (cardiovascular, nervous and skeletal).

transgenic mice↗

NASA NTRSDate not supplied

Overview of the Inaugural Space Health Impacts for the NASA Experience (SHINE) Training Program – Virtual Space Radiation Curriculum

The Space Radiation Element part of NASA Human Research Program initiated a virtual, annual space radiation curriculum. The SHINE space radiation curriculum aims to educate participants not only in the scientific aspects of space radiation but also in the agency’s risk management strategies. SHINE combines weekly seminars by speakers from NASA, other government agencies, academia, and industry, with networking sessions designed to foster collaboration between participants and interactions with NASA scientists. A total of 59 applications were received from which 25 participants were selected. The 25 participants, with citizenship from a total of 8 countries, are composed of 5 graduate students, 4 postdocs, 11 scientists and 5 professors/medical doctors. Participants have a wide range of expertise including molecular and cellular biology, microbiology, botany, physiology, engineering, physics, aerospace medicine, planetary science, biostatistics, and modeling. The inaugural course ran weekly from February 2023 to August 2023 and was comprised of lectures, less formal coffee hours and office hours. The topics covered ranged from space environments to health effects to granting opportunities. The second annual SHINE training program is scheduled from February to August 2024. In addition, a separate competitive practicum session, to allow participants to gain hands-on radiation experience, will be held Fall 2024 at the NASA Space Radiation Laboratory (NSRL).

Janice A. Zawask↗

NASA NTRSDate not supplied

Transcriptomics-based Machine Learning (ML) Analysis Predicts Space-Exposed Murine Livers

NASA has employed high-throughput molecular assays to identify sub-cellular changes impacting human physiology during spaceflight. Machine learning (ML) methods hold the promise to improve our ability to identify important signals within highly dimensional molecular data. However, the inherent limitation of study subject numbers within a spaceflight mission minimizes the utility of ML approaches. To overcome the sample power limitations, data from multiple spaceflight missions must be aggregated while appropriately addressing intra- and inter-study variabilities. Here we describe an approach to log transform, scale and normalize data from six heterogeneous, mouse liver derived transcriptomics datasets (ntotal=137) which enabled ML-methods to perform well (AUC ≥ 0.87) in classifying spaceflown vs ground control animals rather than mission-of-origin. Concordance was found between liver-specific biological processes identified from harmonized ML-based analysis and study-by-study classical omics analysis. This work demonstrates the feasibility of applying ML methods on integrated, heterogeneous datasets of small sample size.

Machine Learning↗

NASA NTRSDate not supplied

Overview of The Inaugural Space Health Impacts for the NASA Experience (SHINE) Training Program – Virtual Space Radiation Curriculum

The Space Radiation Element of the NASA Human Research Program initiated a virtual, annual space radiation curriculum. The Space Health Impacts for the NASA Experience (SHINE) space radiation didactic curriculum aims to educate participants not only in the scientific aspects of space radiation but also in the agency’s risk management strategies. SHINE combines weekly seminars by speakers from NASA, other government agencies, academia, and industry, with networking sessions designed to foster collaboration between participants and interactions with NASA scientists and HRP funded investigators. The inaugural course ran weekly from February 2023 to August 2023 and was comprised of lectures, less formal coffee hours and office hours. The SHINE topics ranged from space environments to health effects and countermeasure to granting opportunities. Participants developed beam time proposals for real, proposed, or potential experiments at the NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory. For the 2023 course, a total of 59 applications were received from which 25 participants were selected. Selected participants were citizens from 8 countries and comprised 5 graduate students, 4 postdocs, 11 scientists and 5 professors/medical doctors. Participants had a wide range of expertise including molecular and cellular biology, microbiology, botany, physiology, engineering, physics, aerospace medicine, planetary science, biostatistics, and modeling. The second annual SHINE training program is scheduled from February to August 2024. In addition, a separate SHINE space radiation practicum session, to allow participants to gain hands-on radiation experience, will be held Fall 2024 at the NSRL.

Radiation↗

NASA NTRSDate not supplied

Overview of The Inaugural Space Health Impacts for the NASA Experience (SHINE) Training Program - Virtual Space Radiation Curriculum

SHINE↗

NASA NTRSDate not supplied

NASA's Space Health Impacts for the NASA Experience (SHINE) Training Program – Space Radiation Curriculum

In February 2023, the Space Radiation Element of the NASA Human Research Program initiated a virtual, annual space radiation curriculum. The Space Health Impacts for the NASA Experience (SHINE) Space Radiation Didactic Curriculum aims to educate participants not only in the scientific aspects of space radiation but also in the agency’s risk management strategies. SHINE Space Radiation Didactic Curriculum combined weekly seminars by speakers from NASA, other government agencies, academia, and industry, with networking sessions designed to foster collaboration between the competitively selected participants as well as interactions with NASA scientists and HRP funded investigators. The inaugural course ran weekly from February 2023 to August 2023 and was comprised of lectures, less formal coffee hours, and office hours. Each two-hour seminar sessions hosted 1-3 presentations on topics which ranged from the space radiation environment to health effects and countermeasure to granting opportunities. All lectures were recorded and will be published on the THREE (The Health Risks of Extraterrestrial Environments) website for public access (https://three.jsc.nasa.gov/). As a course requirement, participants developed individual or collaborative beam time proposals for real, proposed, or potential experiments at the NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory. For the 2023 course, 25 participants were selected from a total of 59 applicants. Selected participants were citizens from 8 countries and comprised 5 graduate students, 4 postdocs, 11 scientists and 5 professors/medical doctors. Participants had a wide range of expertise including molecular and cellular biology, microbiology, botany, physiology, engineering, physics, aerospace medicine, planetary science, biostatistics, and modeling. The second annual SHINE training program is scheduled from February to August 2024. In addition, a separate SHINE Space Radiation Practicum session will be held in Fall 2024 at the NSRL. The SHINE Space Radiation Practicum is a unique opportunity that has not been available to the public since the closure of the NASA Space Radiation Summer School in 2017 and will allow a small cohort of participants competitively selected in Fall 2023 to gain hands-on radiation experience.

SHINE↗

NASA NTRS2018

Leaf Water Status Estimated from Visible-NIR Polarization Measurements

Remotely sensing the water status of plant canopies remains a long term goal of remote sensing research. Existing approaches to remotely sense canopy water status, such as the Crop Water Stress Index (CWSI) and the Equivalent Water Thickness (EWT), have limitations. The CWSI, based upon remotely sensing canopy radiant temperature in the thermal infrared spectral region, does not work well in humid regions, requires estimates of the vapor pressure deficit near the canopy during the remote sensing over-flight and, once stomata close, provides little information regarding the canopy water status. The EWT is based upon the physics of water-light interaction in the 900-2000nm spectral region, not plant physiology. Our goal is development of a remote sensing technique for estimating plant water status based upon measurements in the VIS/NIR spectral region, taking advantage of polarization physics. This technique would potentially provide remote sensing access to plant dehydration physiology – to the changes in cellular photochemistry and structure associated with water deficits in leaves. In this research, we used crossed optical polarization filters to measure the VIS/NIR light reflected from the leaf interior, R, as well as the leaf transmittance, T, for 78 corn (Zea mays) and soybean (Glycine max) leaves having relative water content (RWC) values between 0.60 and 0.98. Our results show that as RWC decreases R increases while T decreases. Our results tie R and T changes in the VIS/NIR to leaf physiological changes – linking the light scattered out of the drying leaf interior to its relative water content and to changes in leaf cellular structure and pigments.

Water↗

DOE OSTI2026

Human RNome Project draft human RNome sequence of GM12878, B-cell line, obtained by mass-spectrometry sequencing, long-read sequencing and short-read sequencing.

Here we report the first draft of the human RNome sequence, a reference map of RNA chemical modifications in a human B-cell line. RNA carries a diverse repertoire of chemical modifications that regulate gene expression, cellular function, and responses to physiological and pathological cues. Yet, unlike the genome, no reference map of RNA modifications is available for any human cell. To generate this resource, the Human RNome Project Consortium analyzed a shared RNA preparation from the well-characterized GM12878 B-cell line using short-read sequencing, long-read direct RNA sequencing, and mass spectrometry, generating more than 7.1 billion sequencing reads spanning approximately 1.2 trillion nucleotides. The resulting maps of the human RNome reveal that RNA modifications are organized according to function, transcript architecture, and cellular identity. Modifications concentrate at functional centers of ribosomal and transfer RNAs, follow the canonical topology of N6-methyladenosine in coding transcripts, and form coordinated hotspots in immune regulatory genes. This first reference human RNome provides a foundation for understanding how RNA chemistry shapes cellular identity, human disease, and the development of RNA-based therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Three-dimensional imaging of mitochondrial cristae complexity using cryo-soft X-ray tomography

Mitochondria are dynamic organelles that change morphology to adapt to cellular energetic demands under both physiological and stress conditions. Cardiomyopathies and neuronal disorders are associated with structure-related dysfunction in mitochondria, but three-dimensional characterizations of the organelles are still lacking. In this study, we combined high-resolution imaging and 3D electron density information provided by cryo-soft X-ray tomography to characterize mitochondria cristae morphology isolated from murine. Using the linear attenuation coefficient, the mitochondria were identified (0.247 ± 0.04 µm -1 ) presenting average dimensions of 0.90 ± 0.20 µm in length and 0.63 ± 0.12 µm in width. The internal mitochondria structure was successfully identified by reaching up the limit of spatial resolution of 35 nm. The internal mitochondrial membranes invagination (cristae) complexity was calculated by the mitochondrial complexity index (MCI) providing quantitative and morphological information of mitochondria larger than 0.90 mm in length. The segmentation to visualize the cristae invaginations into the mitochondrial matrix was possible in mitochondria with MCI ≥ 7. Altogether, we demonstrated that the MCI is a valuable quantitative morphological parameter to evaluate cristae modelling and can be applied to compare healthy and disease state associated to mitochondria morphology.

3-D reconstruction↗

NASA NTRS2001

NO, nitrotyrosine, and cyclic GMP in signal transduction

Over the past 25 years, the role of nitric oxide (NO) in biology has evolved from being recognized as an environmental pollutant to an endogenously produced substance involved in cell communication and signal transduction. NO is produced by a family of enzymes called nitric oxide synthases (NOSs), which can be stimulated by a variety of factors that mediate responses to various stimuli. NO can initiate its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC), or through several other chemical reactions. Activation of sGC results in the production of 3',5'-cyclic guanosine monophosphate (cGMP), an intracellular second messenger signaling molecule, which can subsequently mediate such diverse physiological events such as vasodilatation and immunomodulation. Chemically reactive NO can affect physiological changes through modifications to cellular proteins, one of which is tyrosine nitration. The demonstration that NO is involved in so many biological pathways indicates the importance of this endogenously produced substance, and suggests that there is much more to be discovered about its role in biology in years to come.

Non-NASA Center↗

NASA NTRS2013

Spaceflight and the Mouse Eye: Results from Experiments on Shuttle Missions STS-133 and STS-135

Vision alterations associated with globe flattening, chorodial folds and papilledema, shown in some crew members returning from long duration missions. Hypothesis: Ocular neuroanatomical changes observed in the VIIP syndrome are accompanied by retinal changes at the molecular and cellular level that may affect retinal health and physiology. Objective: Investigate evidence of ocular (retinal) changes associated with spaceflight: (1) histological markers of cellular death and damage (2) molecular markers of oxidative stress (3) gene expression markers of stress

Zanello, Susana B.↗

DOE OSTI2024

Editorial: Cellular heterogeneity in plants

Multicellular eukaryotic organisms, such as plants, consist of various cell types. Despite possessing the same genetic information, each cell exhibits distinct utilization of this information, resulting in the development of unique molecular, physiological, and morphological properties as well as cellular heterogeneity within the organism. This cellular heterogeneity is needed to support plant development and adaptation to environmental changes. Identifying the mechanisms responsible for the differentiation of distinct cell types and precisely characterizing the molecular, biochemical, biophysical, and morphological characteristics of each cell type in various plant species remains a significant objective for plant scientists. Despite the biological importance of these cellular attributes, they have not been adequately described. In this Frontiers Research Topic, “Cellular Heterogeneity in Plants,” various scientific papers provide valuable new insights into the causes and consequences of cellular heterogeneity in plants.

59 BASIC BIOLOGICAL SCIENCES↗