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DOE OSTI2023

Metagenomic stable isotope probing reveals bacteriophage participation in soil carbon cycling

Soil viruses are important components of the carbon (C) cycle, yet we still know little about viral ecology in soils. We added diverse 13 C-labelled carbon sources to soil and we used metagenomic-SIP to detect 13 C assimilation by viruses and their putative bacterial hosts. These data allowed us to link a 13 C-labelled bacteriophage to its 13 C-labelled Streptomyces putative host, and we used qPCR to track the dynamics of the putative host and phage in response to C inputs. Following C addition, putative host numbers increased rapidly for 3 days, and then more gradually, reaching maximal abundance on Day 6. Viral abundance and virus:host ratio increased dramatically over 6 days, and remained high thereafter (8.42 ± 2.94). From Days 6 to 30, virus:host ratio remained high, while putative host numbers declined more than 50%. Putative host populations were 13 C-labelled on Days 3–30, while 13 C-labelling of phage was detected on Days 14 and 30. This dynamic suggests rapid growth and 13 C-labelling of the host fueled by new C inputs, followed by extensive host mortality driven by phage lysis. Furthermore, these findings indicate that the viral shunt promotes microbial turnover in soil following new C inputs, thereby altering microbial community dynamics, and facilitating soil organic matter production.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Pediatric dosing for locally acting drugs in submissions to the U.S. Food and Drug Administration between 2002 and 2020

Abstract Deriving pediatric doses for locally acting drugs (LADs) presents a unique challenge because limited systemic exposure hinders commonly used approaches such as pharmacokinetic matching to adults. This study systematically evaluated drug development practices used for pediatric dose selection of LADs approved by the U.S. Food and Drug Administration from 2002 to 2020. The three study objectives were: (1) to determine the dose selection approach for the labeled pediatric dose, (2) to examine the studied pediatric dose(s), and (3) to evaluate the characteristics of the pediatric clinical programs used to support the labeled pediatric dose. A total of 187 pediatric submissions were characterized for the labeled and studied pediatric doses of LADs. The pediatric dose was predominantly labeled as a flat dose (91%) and at a single‐dose level (67%) similar to adults. The majority (68.4%) of the submissions had the same labeled dose for pediatrics and adults. Independent pharmacodynamic/efficacy studies in pediatric patients commonly (64.2%) provided supportive evidence for the labeled pediatric dose. Inhalation, nasal, and injectable submissions had the highest number of clinical trials, lowest usage of an extrapolation of efficacy approach, and utilized diverse approaches in selecting the studied pediatric doses. This article highlights approaches for LAD dosing in pediatric patients and can be used to inform drug development of these products in the pediatric population.

Research & Experimental Medicine↗

DOE OSTI2020

Differential ultrastructural alterations in the Vglut2 glutamatergic input to the substantia nigra pars compacta/pars reticulata following nigrostriatal dopamine loss in a progressive mouse model of Parkinson’s disease

Abstract Loss of nigrostriatal dopamine (DA) in Parkinson's disease results in over‐activation/bursting of the subthalamic nucleus (STN). The STN projects to the substantia nigra (SN) pars compacta (SNpc) and pars reticulata (SNpr). The vesicular glutamate transporter 2 (Vglut2) is localized within at least STN terminals synapsing within the SN, but it is not known if there are differential changes in the Vglut2+ input to the SNpc versus SNpr following DA loss. The goal/rationale of this current study was to determine whether there were differential changes in the density/levels of glutamate immuno‐gold labeling within Vglut2+ nerve terminals synapsing in the SNpc/SNpr and in the proportion of Vglut2+ terminals contacting tyrosine hydroxylase (TH) positively(+) or negatively(−) labeled dendrites following DA loss. Within the SNpc, there was a significant increase (51.3%) in the density of nerve terminal glutamate immuno‐gold labeling within Vglut2+ terminals synapsing on TH(−) dendrites following MPTP versus the vehicle ( VEH ) group. There was a significant decrease (16%) in the percentage of Vglut2+ terminals contacting TH(+) labeled dendrites in the MPTP‐ versus VEH‐treated group within the SNpc. Within the SNpr, there was a significant decrease in the density of glutamate immuno‐gold labeling in Vglut2+ terminals contacting TH(+) (71.5%) and TH(−) (55.5%) labeled dendrites, suggesting an increase in glutamate release. There was no change in the percentage of Vglut2+ terminals contacting TH(+) or TH(−) dendrites in the SNpr. We conclude that there is a differential effect following DA loss on the glutamate input from Vglut2+ terminals synapsing within the SNpr versus SNpc.

Moore, Cynthia↗

DOE OSTI2024

Mycorrhizal associations of temperate forest seedlings mediate rhizodeposition, but not soil carbon storage, under elevated nitrogen availability

Abstract Tree‐mycorrhizal associations are associated with patterns in nitrogen (N) availability and soil organic matter storage; however, we still lack a mechanistic understanding of what tree and fungal traits drive these patterns and how they will respond to global changes in soil N availability. To address this knowledge gap, we investigated how arbuscular mycorrhizal (AM)‐ and ectomycorrhizal (EcM)‐associated seedlings alter rhizodeposition in response to increased seedling inorganic N acquisition. We grew four species each of EcM and AM seedlings from forests of the eastern United States in a continuously 13 C‐labeled atmosphere within an environmentally controlled chamber and subjected to three levels of 15 N‐labeled fertilizer. We traced seedling 15 N uptake from, and 13 C‐labeled inputs (net rhizodeposition) into, root‐excluded or ‐included soil over a 5‐month growing season. N uptake by seedlings was positively related to rhizodeposition for EcM‐ but not AM‐associated seedlings in root‐included soils. Despite this contrast in rhizodeposition, there was no difference in soil C storage between mycorrhizal types over the course of the experiment. Instead root‐inclusive soils lost C, while root‐exclusive soils gained C. Our findings suggest that mycorrhizal associations mediate tree belowground C investment in response to inorganic N availability, but these differences do not affect C storage. Continued soil warming and N deposition under global change will increase soil inorganic N availability and our seedling results indicate this could lead to greater belowground C investment by EcM‐associated trees. This potential for less efficient N uptake by EcM‐trees could contribute to AM‐tree success and a shift toward more AM‐dominated temperate forests.

Fitch, Amelia A.↗

DOE OSTI2020

Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria ( Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (C i ), but some evidence indicates that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of C i incorporation into different taxa, mat samples were incubated with [ 13 C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of 13 C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with 13 C. Most 13 C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins associated with photosynthesis, C i fixation, and N 2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for C i fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to C i fixation. Other taxa showed much lower [ 13 C]bicarbonate incorporation. IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven C i fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in C i fixation. Because this pathway imparts a lower degree of selection of isotopically heavy C i than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of 13 C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the 13 C/ 12 C ratios of mat biomass will help geochemists interpret the 13 C/ 12 C ratios of organic carbon in the fossil record.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes

Abstract Background Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput. Results Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software ( https://sourceforge.net/projects/calis-p/ ). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using 18 O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics. Conclusions We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Underwater Target Detection Software Demonstration on the RivGen Turbine

This repository contains data and processing scripts necessary to train the object detection models utilized in the underwater target detection software demonstration on the RivGen turbine project and to produce performance metrics (precision, recall, mAP50, mAP50-95). - Contents - Data consist of "images" and "labels". Each image has an associated label, both share the same time string in its file name (e.g., 2024_05_25_09_01_57.98.jpg and 2024_05_25_09_01_57.98.txt). Time strings have the format %yyyy_%mm_%dd_%HH_%MM_%SS.%3f. Images and labels were curated from 2021 and 2024 smolt outmigration periods at the project site in Igiugig, AK. Images are monochrome 8-bit images of objects (smolt, debris, and other) passing through the field of view of the deployed cameras during various operational stages of the RivGen turbine. Labels are text files indicating the class and bounding polygon of each object in an image. The provided labels use the "YOLO" label format. - Requirements - Python3.8+ is required to install and run the train and validation script. The README.md provides instruction for installing the requirements from the requirements.py file. - Instructions - The "example_train.py" file ingests the provided data, trains a model, and produces model performance metrics at completion. NOTE: model performance metrics will vary from run to run as a consequence of the random selection of training and validation data.

16 TIDAL AND WAVE POWER↗

DOE OSTI2026

Custom-trained Machine-learning Interatomic Potentials: ZnCl2 Aqueous Solution

This dataset was generated using an iterative active-learning strategy implemented in the ArcaNN software package (https://github.com/arcann-chem/arcann_training) to train machine-learning interatomic potentials for aqueous ZnCl2 solutions. Each active-learning cycle consisted of three stages: training, exploration, and labeling. The initial training set combined configurations generated in this work from enhanced-sampling ab initio molecular dynamics simulations with configurations from a previously reported neural-network-potential study of aqueous ZnCl2. The enhanced-sampling ab initio molecular dynamics simulations involved Zn–Cl separation and the chloride coordination number around Zn²? as collective variables. These configurations served as the seed dataset. Subsequent active-learning cycles expanded the training set by identifying and labeling configurations that were poorly represented by the current models, thereby improving coverage of ion-association states and changes in local coordination and charge-state environments relevant to the solution free-energy landscape. For all selected configurations, single-point calculations of the total energies and atomic forces were performed within density functional theory using the CP2K Quickstep module. Reference calculations employed the revPBE-D3 and r2SCAN exchange-correlation functionals. Motivated by recent work on aqueous Zn²?, the main revPBE calculations omitted D3 dispersion contributions involving Zn²?, while retaining the D3 correction for water and chloride. For comparison, fully dispersion-corrected revPBE-D3 reference calculations were also performed, with D3 applied to all species, including Zn²?. Valence electrons were treated explicitly, while core electrons were represented using norm-conserving Goedecker–Teter–Hutter pseudopotentials. The wave functions were expanded using the mixed Gaussian-and-plane-wave scheme with TZV2P-MOLOPT basis sets for all elements and a 600 Ry auxiliary plane-wave cutoff for the electron density. Self-consistent-field convergence was accelerated using the orbital-transformation and Direct Inversion in the Iterative Subspace algorithms, with a convergence threshold of 10?6. All single-point calculations were performed in periodic orthorhombic cells. The CELL_REF keyword in CP2K was used to define a fixed reference cell with a box length of 25 Å. This treatment ensured a consistent reference for configurations extracted from NpT trajectories with fluctuating cell dimensions. The resulting DFT energies and atomic forces constitute the ground-truth labels used to train the MLIPs. The resulting MLIP was trained for aqueous ZnCl2 solutions spanning concentrations from 0 to 30 molal and a broad pH range, from strongly acidic to strongly basic conditions. Representative examples of configurations included in the MLIP training dataset are provided below. These include 1) Representative configurations from the dataset labeled at the revPBE-D3 level, with D3 dispersion interactions involving Zn2+ excluded (revPBE-wo-D3). 2) Representative configurations from the dataset labeled at the fully dispersion-corrected revPBE-D3 level, with D3 interactions applied to all species, including Zn2+ (revPBE-D3). 3) Representative configurations from the dataset labeled at the r2SCAN level of theory (r2SCAN).

Dinpajooh, Mohammadhasan [Pacific Northwest Nation↗

NASA NTRS1976

DHARMA - Discriminant hyperplane abstracting residuals minimization algorithm for separating clusters with fuzzy boundaries

Learning of discriminant hyperplanes in imperfectly supervised or unsupervised training sample sets with unreliably labeled samples along the fuzzy joint boundaries between sample clusters is discussed, with the discriminant hyperplane designed to be a least-squares fit to the unreliably labeled data points. (Samples along the fuzzy boundary jump back and forth from one cluster to the other in recursive cluster stabilization and are considered unreliably labeled.) Minimization of the distances of these unreliably labeled samples from the hyperplanes does not sacrifice the ability to discriminate between classes represented by reliably labeled subsets of samples. An equivalent unconstrained linear inequality problem is formulated and algorithms for its solution are indicated. Landsat earth sensing data were used in confirming the validity and computational feasibility of the approach, which should be useful in deriving discriminant hyperplanes separating clusters with fuzzy boundaries, given supervised training sample sets with unreliably labeled boundary samples.

Dasarathy, B. V.↗

NASA NTRS1988

A hardware implementation of a relaxation algorithm to segment images

Relaxation labelling is a mathematical technique frequently applied in image processing algorithms. In particular, it is extensively used for the purpose of segmenting images. The paper presents a hardware implementation of a segmentation algorithm, for images consisting of two regions, based on relaxation labelling. The algorithm determines, for each pixel, the probability that it should be labelled as belonging to a particular region, for all regions in the image. The label probabilities (labellings) of every pixel are iteratively updated, based on those of the pixel's neighbors, until they converge. The pixel is then assigned to the region correspondent to the maximum label probability. The system consists of a control unit and of a pipeline of segmentation stages. Each segmentation stage emulates in the hardware an iteration of the relaxation algorithm. The design of the segmentation stage is based on commercially available digital signal processing integrated circuits. Multiple iterations are accomplished by stringing stages together or by looping the output of a stage, or string of stages, to its input. The system interfaces with a generic host computer. Given the modularity of the architecture, performance can be enhanced by merely adding segmentation stages.

Loda, Antonio G.↗

NASA NTRS1991

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins E2 and F2(alpha) which regulate protein turnover rates and muscle cell growth. Mechnical stimulation significantly increases the breakdown rate of (3)H-arachidonic acid labelled phospholipids, releasing free (3)H-arachidonic acid, and the rate-limiting precursor of prostaglandin synthesis. Mechanical stimulation also significantly increases (3)H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-2-(3)H inositol labelled phospholipids. Phospholipase A2, phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are activated by stretch. The lipase inhibitors bromophenacylbromide and RHC80267 together reduce stretch-induced prostaglandin production by 73-83 percent. The stretch-induced increases in prostaglandin production, (3)H-arachidonic acid labelled phospholipid breakdown, and (3)H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-2-(3)H inositol labelled phospholipids are dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and prostaglandins through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

Vandenburgh, Herman H.↗

NASA NTRS1993

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E2 and F2 alpha which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of 3H-arachidonic acid labelled phospholipids, releasing free 3H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases 3H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-3H]inositol labelled phospholipids. Phospholipase A2 (PLA2), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, 3H-arachidonic acid labelled phospholipid breakdown, and 3H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-[2-3H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

NASA Discipline Number 40-40↗

NASA NTRS2006

Fluorescent Applications to Crystallization

By covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and tests with model proteins have shown that labeling u to 5 percent of the protein molecules does not affect the X-ray data quality obtained . The presence of the trace fluorescent label gives a number of advantages. Since the label is covalently attached to the protein molecules, it "tracks" the protein s response to the crystallization conditions. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a darker background. Non-protein structures, such as salt crystals, do not show up under fluorescent illumination. Crystals have the highest protein concentration and are readily observed against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. Preliminary tests, using model proteins, indicates that we can use high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that more rapid amorphous precipitation kinetics may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Experiments are now being carried out to test this approach using a wider range, of proteins. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons.

Pusey, Marc L.↗

NASA NTRS1980

Cell sorting apparatus

Polymeric functional microspheres containing metal or metal compounds are formed by addition polymerization of a covalently bondable olefinic monomer such as hydroxyethylmethacrylate in the presence of finely divided metal or metal oxide particles, such as iron, gold, platinum or magnetite, which are embedded in the resulting microspheres. The microspheres can be covalently bonded to chemotherapeutic agents, antibodies, or other proteins providing a means for labeling or separating labeled cells. Labeled cells or microspheres can be concentrated at a specific body location such as in the vicinity of a malignant tumor by applying a magnetic field to the location and then introducing the magnetically attractable microspheres or cells into the circulatory system of the subject. Labeled cells can be separated from a cell mixture by applying a predetermined magnetic field to a tube in which the mixture is flowing. After collection of the labeled cells, the magnetic field is discontinued and the labeled sub-cell population recovered.

Yen, Shiao-Ping S.↗

NASA NTRS1980

Method for producing a biological reagent

Yen, Shiao-Ping S.↗

NASA NTRS1999

Real-time biochemical assay telemetering system

The present invention is an apparatus and a method of detecting a chemical released by perspiration, typically through sweat and broadcasting the detection to a receiver. The chemical may be a drug of abuse. The device which is attached to the skin of a subject contains labeled antibodies or label containing microspheres attached to antibodies. The labeled antibodies are bound to solid phase drug via antigen-antibody interaction. These labeled antibodies are displaced from the solid phase support to which they are bound by free drug molecules in the perspiration. These labeled antibodies then migrate through a spacer layer and are trapped by a layer containing a suitable selective binding material. The label is illuminated or excited by a light source and detected by a photodetector. The signal can be recorded, or transmitted to a remote radio monitor.

Kidwell, David A.↗

NASA NTRS1979

Metal containing polymeric functional microspheres

Yen, Shiao-Ping S.↗

NASA NTRS1999

Decal Process Document and Catalog

The Decal Process Document and Catalog, JSC 27260 is the standard flight decal catalog, complete with illustrations and part numbers. As hardware developers identify labels that have common applicability across end items, these labels can be evaluated for "standard decal classification" and entered into the decal catalog for general use. The hardware developer must have a label design that meets current, applicable labeling requirements, and submit to the Decal Design and Production Facility (DDPF) as a standard label candidate. Upon approval, the label will be added to the decal catalog. The Decal Process Document and Catalog provides a selection of decals from which the NASA and NASA contractor customers can easily order. The decals shown in the catalog have been previously produced and have released engineering/fabrication drawings on file in the (DDPF). A released drawing is required before a decal can be produced or placed into the catalog. Some decals included in the catalog have a common applicability and are used in various NASA vehicles/habitats. It is the intent of the DDPF to maintain this catalog as a "living document" to which decals/placards can be added as they are repeatedly used. The advantage of identifYing flight decals in this catalog is that a released drawing is already in place, and the products will be flight certified.

Source record↗