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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 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Molecularly Imprinted Polymer Sensor Empowered by Bound States in the Continuum for Selective Trace-Detection of TGF-beta

The integration of advanced materials and photonic nanostructures can lead to enhanced biodetection capabilities, crucial in clinical scenarios and point-of-care diagnostics, where simplified strategies are essential. Herein, a molecularly imprinted polymer (MIP) photonic nanostructure is demonstrated, which selectively binding to transforming growth factor-beta (TGF-β), in which the sensing transduction is enhanced by bound states in the continuum (BICs). The MIP operating as a synthetic antibody matrix and coupled with BIC resonance, enhances the optical response to TGF-β at imprinted sites, leading to an augmented detection capability, thoroughly evaluated through spectral shift and optical lever analogue readout. The validation underscores the MIP-BIC sensor capability to detect TGF-β in spiked saliva, achieving a limit of detection of 10 fM and a resolution of 0.5 pM at physiological concentrations, with a precision of two orders of magnitude above discrimination threshold in patients. The MIP tailored selectivity is highlighted by an imprinting factor of 52, showcasing the sensor resistance to interference from other analytes. The MIP-BIC sensor architecture streamlines the detection process eliminating the need for complex sandwich immunoassays and demonstrates the potential for high-precision quantification. This positions the system as a robust tool for biomarker detection, especially in real-world diagnostic scenarios.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2025

Human IgE monoclonal antibodies define two unusual epitopes trapping dog allergen Can f 1 in different conformations

Abstract Molecular analysis of interactions between IgE antibody and allergen allows the structural basis of IgE recognition to be defined. Human IgE (hIgE) epitopes of respiratory lipocalin allergens, including Can f 1, remain elusive due to a lack of IgE‐allergen complexes. This study aims to map the structure of allergenic epitopes on Can f 1. The fragment antigen‐binding (Fab) regions of Can f 1 specific human IgE monoclonal antibodies (hIgE mAb) were used to determine the structures of IgE epitopes. Epitope mutants were designed to target Can f 1 epitopes. Immunoassays and a human FcεRIαtransgenic mouse model of passive anaphylaxis in vivo were used to assess the functional activity of epitope mutants. Crystal structures of natural or recombinant Can f 1 complexed with two hIgE mAb 1J11 and 12F3 Fabs, respectively, were determined. The hIgE mAb bound to two partially overlapping epitopes and recognized two different Can f 1 conformations. The hIgE mAb 12F3 showed an unusual mode of binding by protruding its heavy chain CDR3 inside the Can f 1 calyx. Epitope mutants generated based on the structural analyses displayed a 64%–89% reduction in IgE antibody binding and failed to induce passive anaphylaxis in a human FcεRIαtransgenic mouse model. In summary, the structures of Can f 1‐hIgE Fab complexes revealed two unique and partially overlapping epitopes on Can f 1. The modification of the identified IgE epitopes provides a pathway for the design of hypoallergens to treat dog allergies.

Biochemistry & Molecular Biology↗
DOE OSTI2026

High-Sensitivity DNA Aptasensors for Detecting Salivary Biomarker S100A7 in Heart Failure

Early detection of heart failure (HF) is vital for improving patient outcomes, lowering hospital readmission rates, and enabling prompt treatment. We present the first high-affinity DNA aptamer for the salivary HF biomarker S100A7 and its application in highly sensitive, noninvasive diagnostic tests. Iterative truncation of the initial 82-nt aptamer (17–82) produced a 43-nt core (17–43) with a binding affinity of 27 nM, which was further enhanced to 5.5 nM through dimerization. Biochemical and mutational studies confirmed that 17–43 adopts a G-quadruplex structure, which is essential for S100A7 recognition and resistance to enzymatic degradation in human saliva. Incorporating 17–43 into sandwich aptamer-ELISA and hybrid aptamer–antibody ELISA assays allowed detection of recombinant S100A7 in human saliva with limits of detection (LOD) of 7.4 ng mL–1 (0.6 nM) and 29 pg/mL (2.2 pM), respectively-outperforming commercial immunoassays in both sensitivity and dynamic range. The hybrid assay maintained its full performance after 2.5 months of room temperature storage. Additionally, a biolayer interferometry (BLI) sensor with 17–43 quantified S100A7 in patient saliva (n = 3), achieving a LOD of 3.2 ng mL–1 (0.3 nM) with a total assay time of less than 20 min. The aptamer’s stability, high specificity, and versatility across biosensing platforms establish it as a promising tool for noninvasive heart failure diagnostics, laying the groundwork for portable aptamer-based biosensors for multiplexed monitoring of HF biomarkers.

DNA aptamer↗
DOE OSTI2021

Nickel–Platinum Nanoparticles as Peroxidase Mimics with a Record High Catalytic Efficiency

While nanoscale mimics of peroxidase have been extensively developed over the past decade or so, their catalytic efficiency as a key parameter has not been substantially improved in recent years. Herein, we report a class of highly efficient peroxidase mimic–nickel–platinum nanoparticles (Ni–Pt NPs) that consist of nickel-rich cores and platinum-rich shells. The Ni–Pt NPs exhibit a record high catalytic efficiency with a catalytic constant (K cat ) as high as 4.5 × 10 7 s –1 , which is ~46- and 10 4 -fold greater than the K cat values of conventional Pt nanoparticles and natural peroxidases, respectively. Density functional theory calculations reveal that the unique surface structure of Ni–Pt NPs weakens the adsorption of key intermediates during catalysis, which boosts the catalytic efficiency. Furthermore, the Ni–Pt NPs were applied to an immunoassay of a carcinoembryonic antigen that achieved an ultralow detection limit of 1.1 pg/mL, hundreds of times lower than that of the conventional enzyme-based assay.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Morphine and high-fat diet differentially alter the gut microbiota composition and metabolic function in lean versus obese mice

There are known associations between opioids, obesity, and the gut microbiome, but the molecular connection/mediation of these relationships is not understood. To better clarify the interplay of physiological, genetic, and microbial factors, this study investigated the microbiome and host inflammatory responses to chronic opioid administration in genetically obese, diet-induced obese, and lean mice. Samples of feces, urine, colon tissue, and plasma were analyzed using targeted LC-MS/MS quantification of metabolites, immunoassays of inflammatory cytokine levels, genome-resolved metagenomics, and metaproteomics. Genetic obesity, diet-induced obesity, and morphine treatment in lean mice each showed increases in distinct inflammatory cytokines. Metagenomic assembly and binning uncovered over 400 novel gut bacterial genomes and species. Morphine administration impacted the microbiome’s composition and function, with the strongest effect observed in lean mice. This microbiome effect was less pronounced than either diet or genetically driven obesity. Based on inferred microbial physiology from the metaproteome datasets, a high-fat diet transitioned constituent microbes away from harvesting diet-derived nutrients and towards nutrients present in the host mucosal layer. Considered together, these results identified novel host-dependent phenotypes, differentiated the effects of genetic obesity versus diet induced obesity on gut microbiome composition and function, and showed that chronic morphine administration altered the gut microbiome.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ~2500 proteins and precise quantification of ~1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Implementation of rapid diagnostics assays for detection of histoplasmosis and cryptococcosis in central american people living with HIV

Abstract Objectives Histoplasmosis and cryptococcosis are important public health problems in people living with HIV (PLHIV) in Central America. Conventional laboratory assays, based on microscopy and culture, are not optimal for the diagnosis of either disease. However, antigen (Ag) assays are rapid and highly accurate for the diagnosis of these infections. Methods Laboratory surveillance of PLHIV was carried out in four hospitals in Panama, Honduras and Nicaragua, between 2015 and 2019. Detection of Histoplasma antigens in urine was performed by enzyme immunoassay (EIA), and Cryptococcus antigen detection in sera and cerebrospinal fluid specimens was performed by lateral flow assay (LFA). Results A total of 4,453 PLHIV with clinical suspicion of histoplasmosis ( n = 1,343) or cryptococcosis ( n = 3,110; 2,721 sera and 389 CSF) were tested. Of 1,343 patients suspected of having histoplasmosis, 269 (20%) were Histoplasma Ag positive. Of 3,110 patients tested using the Cryptococcus Ag assay, 329 (11%) were positive. Honduras reported the highest positivity rates (32% for Histoplasma Ag, and 16% for Cryptococcus Ag); Panama reported the largest number of patients testing positive using the Histoplasma Ag assay ( n = 201); and Nicaragua reported the largest number of patients testing positive using the Cryptococcus Ag assay ( n = 170). Conclusion Here, we show how the implementation of rapid diagnostics assays impacted case detection and was useful for the care of people with advanced HIV. Rapid and accurate diagnosis could reduce mortality associated with histoplasmosis and cryptococcosis in PLHIV.

Caceres, Diego H.↗
DOE OSTI2022

Composition and yield of non-cellulosic and cellulosic sugars in soluble and particulate fractions during consolidated bioprocessing of poplar biomass by Clostridium thermocellum

Abstract Background Terrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed. Results The total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I. Conclusions The significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum .

09 BIOMASS FUELS↗
DOE OSTI2025

Biomarkers, Proteoforms, and Mass Spectrometry–Based Assays for Diabetes Clinical Research

Abstract The prevalence of diabetes, particularly type 2 diabetes, has reached epidemic proportions globally. The number of patients with type 1 diabetes (T1D) is also increasing rapidly. Despite advancements in understanding the pathogenesis of diabetes, the lack of circulating pancreatic biomarkers and reliable clinical-grade assays remains a major gap in diabetes research, often hindering the ability to adequately assess disease progression and therapeutic responses. This mini-review discusses emerging pancreatic biomarkers, with an emphasis on T1D, the limitations of current immunoassays, and the expanding role of mass spectrometry–based assays. Highlights include the recent work within the NIDDK-funded “Targeted Mass Spectrometry Assays for Diabetes and Obesity Research (TaMADOR)” consortium, which aims to develop robust, quantitative, and transferable assays for translational research. The review also emphasizes the importance of proteoform-specific assays for monitoring pancreatic function, including prohormone processing during disease progression or in responses to therapy.

Endocrinology & Metabolism↗
DOE OSTI2024

Temporal serum neurofilament light chain concentrations in sheep inoculated with the agent of classical scrapie

Objective: Neurofilament light chain (Nf-L) has been used to detect neuroaxonal damage in the brain caused by physical injury or disease. The purpose of this study was to determine if serum Nf-L could be used as a biomarker for pre-symptomatic detection of scrapie in sheep. Methods: Four sheep with prion protein genotype AVQQ were intranasally inoculated with the classical scrapie strain x124. Blood was collected every 4 weeks until 44 weeks post-inoculation, at which point weekly collection commenced. Serum was analyzed using single molecule array (Quanterix SR-X) to evaluate Nf-L concentrations. Results: Scrapie was confirmed in each sheep by testing homogenized brainstem at the level of the obex with a commercially available enzyme immunoassay. Increased serum Nf-L concentrations were identified above the determined cutoff during the last tenth of the respective incubation period for each sheep. Throughout the time course study, PrP Sc accumulation was not detected antemortem by immunohistochemistry in rectal tissue at any timepoint for any sheep. RT-QuIC results were inconsistently positive throughout the timepoints tested for each sheep; however, each sheep had at least one timepoint detected positive. When assessing serum Nf-L utility using receiver operator characteristic curves against different clinical parameters, such as asymptomatic and symptomatic (pruritus or neurologic signs), results showed that Nf-L was most useful at being an indicator of disease only late in disease progression when neurologic signs were present. Conclusion: Serum Nf-L concentrations in the cohort of sheep increased as disease progressed; however, serum Nf-L did not increase during the presymptomatic window. The levels increased substantially throughout the final 10% of the animals’ scrapie incubation period when other clinical signs were present. Serum Nf-L is not a reliable biomarker for pre-clinical detection of scrapie.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Disease phenotype of classical sheep scrapie is changed upon experimental passage through white-tailed deer

Prion agents occur in strains that are encoded by the structure of the misfolded prion protein (PrP Sc ). Prion strains can influence disease phenotype and the potential for interspecies transmission. Little is known about the potential transmission of prions between sheep and deer. Previously, the classical US scrapie isolate (No.13-7) had a 100% attack rate in white-tailed deer after oronasal challenge. The purpose of this study was to test the susceptibility of sheep to challenge with the scrapie agent after passage through white-tailed deer (WTD scrapie). Lambs of various prion protein genotypes were oronasally challenged with WTD scrapie. Sheep were euthanized and necropsied upon development of clinical signs or at the end of the experiment (72 months post-inoculation). Enzyme immunoassay, western blot, and immunohistochemistry demonstrated PrP Sc in 4 of 10 sheep with the fastest incubation occurring in VRQ/VRQ sheep, which contrasts the original No.13-7 inoculum with a faster incubation in ARQ/ARQ sheep. Shorter incubation periods in VRQ/VRQ sheep than ARQ/ARQ sheep after passage through deer was suggestive of a phenotype change, so comparisons were made in ovinized mice and with sheep with known strains of classical sheep scrapie: No. 13–7 and x-124 (that has a more rapid incubation in VRQ/VRQ sheep). After mouse bioassay, the WTD scrapie and x-124 isolates have similar incubation periods and PrP Sc conformational stability that are markedly different than the original No. 13–7 inoculum. Furthermore, brain tissues of sheep with WTD scrapie and x-124 scrapie have similar patterns of immunoreactivity that are distinct from sheep with No. 13–7 scrapie. Multiple lines of evidence suggest a phenotype switch when No. 13–7 scrapie prions are passaged through deer. This represents one example of interspecies transmission of prions resulting in the emergence or selection of new strain properties that could confound disease eradication and control efforts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Amplification-free nucleic acid detection with a fluorescence-based waveguide biosensor

Early detection of pathogens using nucleic acids in clinical samples often requires sensitivity at the single-copy level, which currently necessitates time-consuming and expensive nucleic acid amplification. Here, we describe 1) a redesigned flow cell in the shape of a trapezoid-subtracted geometric stadium, and 2) modified experimental procedures that allow for the measurement of sub-attomolar analytes in microliter quantities on a fluorescence-based waveguide biosensor. We verified our instrumental sensitivity with a 200-μL sample of a fluorescent streptavidin conjugate at 100 zM (100 zeptomolar, or 100·10 −21 mol L −1 ) and theoretically explored the applicability of this modified sensing platform in a sandwich immunoassay format using a Langmuir adsorption model. We present assays that demonstrate specific detection of synthetic influenza A DNA (in buffer) and RNA (in saliva) oligonucleotides at the single-copy level (200 μL at 10 zM) using a fluorescent molecular beacon. Lastly, we demonstrate detection of isolated genomic influenza A RNA at a clinically relevant concentration. This work constitutes a sensitivity improvement of over twelve orders of magnitude compared to our previous nucleic acid detection work, illustrating the significant enhancements that can be gained with optimized experimental design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Short incubation periods of atypical H-type BSE in cattle with EK211 and KK211 prion protein genotypes after intracranial inoculation

In 2006, a case of atypical H-type BSE (H-BSE) was found to be associated with a germline mutation in the PRNP gene that resulted in a lysine substitution for glutamic acid at codon 211 (E211K). The E211K amino acid substitution in cattle is analogous to E200K in humans, which is associated with the development of genetic Creutzfeldt-Jakob disease (CJD). In the present study, we aimed to determine the effect of the EK211 prion protein genotype on incubation time in cattle inoculated with the agent of H-BSE; to characterize the molecular profile of H-BSE in KK211 and EK211 genotype cattle; and to assess the influence of serial passage on BSE strain. Eight cattle, representing three PRNP genotype groups (EE211, EK211, and KK211), were intracranially inoculated with the agent of H-BSE originating from either a case in a cow with the EE211 prion protein genotype or a case in a cow with E211K amino acid substitution. All inoculated animals developed clinical disease; post-mortem samples were collected, and prion disease was confirmed through enzyme immunoassay, anti-PrP Sc immunohistochemistry, and western blot. Western blot molecular analysis revealed distinct patterns in a steer with KK211 H-BSE compared to EK211 and EE211 cattle. Incubation periods were significantly shorter in cattle with the EK211 and KK211 genotypes compared to the EE211 genotype. Inoculum type did not significantly influence the incubation period. This study demonstrates a shorter incubation period for H-BSE in cattle with the K211 genotype in both the homozygous and heterozygous forms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Targeted Quantification of Protein Phosphorylation and Its Contributions towards Mathematical Modeling of Signaling Pathways

Post-translational modifications (PTMs) are key regulatory mechanisms that can control protein function. Of these, phosphorylation is the most common and widely studied. Because of its importance in regulating cell signaling, precise and accurate measurements of protein phosphorylation across wide dynamic ranges are crucial to understanding how signaling pathways function. Although immunological assays are commonly used to detect phosphoproteins, their lack of sensitivity, specificity, and selectivity often make them unreliable for quantitative measurements of complex biological samples. Recent advances in Mass Spectrometry (MS)-based targeted proteomics have made it a more useful approach than immunoassays for studying the dynamics of protein phosphorylation. Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—and parallel reaction monitoring (PRM) can quantify relative and absolute abundances of protein phosphorylation in multiplexed fashions targeting specific pathways. In addition, the refinement of these tools by enrichment and fractionation strategies has improved measurement of phosphorylation of low-abundance proteins. The quantitative data generated are particularly useful for building and parameterizing mathematical models of complex phospho-signaling pathways. Potentially, these models can provide a framework for linking analytical measurements of clinical samples to better diagnosis and treatment of disease.

mathematical modeling↗
DOE OSTI2022

Transmission of Raccoon-Passaged Chronic Wasting Disease Agent to White-Tailed Deer

The transmission characteristics of prion diseases are influenced by host prion protein sequence and, therefore, the host species. Chronic wasting disease (CWD), a prion disease of cervids, has widespread geographical distribution throughout North America and occurs in both wild and farmed populations. CWD prions contaminate the environment through scattered excrement and decomposing carcasses. Fresh carcasses with CWD prions are accessible by free-ranging mesopredators such as raccoons and may provide a route of exposure. Previous studies demonstrated the susceptibility of raccoons to CWD from white-tailed deer. In this study, we demonstrate that white-tailed deer replicate raccoon-passaged CWD prions which results in clinical disease similar to intraspecies CWD transmission. Six white-tailed deer were oronasally inoculated with brain homogenate from a raccoon with CWD. All six deer developed clinical disease, had widespread lymphoid distribution of misfolded CWD prions (PrP Sc ), and had neuropathologic lesions with PrP Sc accumulation in the brain. The presence of PrP Sc was confirmed by immunohistochemistry, enzyme-linked immunoassay, and western blot. The western blot migration pattern of raccoon-passaged CWD was different from white-tailed deer CWD. Transmission of raccoon CWD back to white-tailed deer resulted in an interposed molecular phenotype that was measurably different from white-tailed deer CWD.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. In this study, we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Sixty days of head-down tilt bed rest with or without artificial gravity do not affect the neuromuscular secretome

Highlights: • Long-term bed rest with/without artificial gravity does not affect the neuromuscular secretome. • Serum concentrations of markers of muscle wasting are not affected by bed rest/artificial gravity. • C-terminal Agrin Fragment serum concentrations are not affected by bed rest/artificial gravity. • Human centrifugation during bed rest does not have a negative impact on the neuromuscular secretome. Artificial gravity is a potential countermeasure to attenuate effects of weightlessness during long-term spaceflight, including losses of muscle mass and function, possibly to some extent attributable to disturbed neuromuscular interaction. The 60-day AGBRESA bed-rest study was conducted with 24 participants (16 men, 8 women; 33 ± 9 years; 175 ± 9 cm; 74 ± 10 kg; 8 control group, 8 continuous (cAG) and 8 intermittent (iAG) centrifugation) to assess the impact of bed rest with or without daily 30-min continuous/intermittent centrifugation with 1G at the centre of mass. Fasting blood samples were collected before and on day 6, 20, 40 and 57 during 6° head-down tilt bed rest. Concentrations of circulating markers of muscle wasting (GDF-8/myostatin; slow skeletal muscle troponin T; prostaglandin E2), neurotrophic factors (BDNF; GDNF) and C-terminal Agrin Fragment (CAF) were determined by ELISAs. Creatine kinase activity was assessed by colorimetric enzyme assay. Repeated-measures ANOVAs were conducted with TIME as within-subject, and INTERVENTION and SEX as between-subject factors. The analyses revealed no significant effect of bed rest or sex on any of the parameters. Continuous or intermittent artificial gravity is a safe intervention that does not have a negative impact of the neuromuscular secretome.

60 APPLIED LIFE SCIENCES↗