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At least 631 records · Page 35
All sourcesNASA NTRSDOE OSTI
NASA NTRSDate not supplied

Coupling Online Conductivity with Offline Ion Chromatography Measurements using the Particle-into-Liquid Sampler

A particle-into-liquid sampler has been combined with a flow through conductivity cell to provide a continuous, non-destructive, online measurement in support of offline ion chromatography analysis. The conductivity measurement provides a rapid assessment of the total ion concentration augmenting the slower batch data from the offline analysis and is developed primarily to assist airborne measurements, where fast time response is essential. A model of the conductivity was developed for measured ions and excellent closure is derived for laboratory-generated aerosols. The PILS-conductivity measurement was extensively tested during the NASA Cloud, Aerosol and Monsoon Processes: Philippines Experiment (CAMP2Ex) across nineteen research flights and the conductivity data was found to augment the temporal capability of the PILS instrument for assessing ionic species, allowing sub-minute variability to be resolved. Through the sampling of a diverse range of ambient aerosol, including biomass burning, fresh and aged urban pollution, the conductivity measurement offered additional useful information to untangle the composition of complex aerosol mixtures, specifically for assessing acidic aerosol conditions.

Ewan Colin Crosbie↗
DOE OSTI2020

Multiscale investigations of europium(III) complexation with tetra- n -octyl diglycolamide confined in porous solid supports

The microscopic, short-range coordination environments and mesoscopic, long-range structures of Eu 3+ contacted with tetra-n-octyl diglycolamide (TODGA) confined on ordered mesoporous carbon (OMC) nanoparticles and Amberchrom CG 71 resin were probed using Eu L 3 -edge extended X ray absorption fine structure (EXAFS) as well as small-angle and wide-angle X ray scattering, SAXS and WAXS, respectively. A homoleptic Eu(TODGA) 3 3+ coordination complex typical of liquid-liquid extraction (LLE) chemistry is present under low Eu 3+ loading conditions on both solid supports. Deviations from this traditional structure motif appear at hyperstoichiometric Eu 3+ loadings, above the 1:3 Eu 3+ to TODGA mole ratio. Microcrystalline-like domains with multinuclear Eu speciation are templated by use of these high Eu 3+ loading conditions with the functionalized OMC materials, highlighting a major departure of liquid-solid extraction chromatography (EXC) from the coordination chemistry of LLE. No such long-range spatial coherence was observed for analogous polyacrylic resin materials. Furthermore, these results demonstrate both the similarities and differences between multiscale structures in LLE and EXC, underscoring the opportunities for improved separation techniques based on solid supports with long-range spatial coherence (e.g., OMC systems) and without it (e.g., resin materials). Since crystallization was the first approach to adjacent lanthanide separations, the prospect of templating microcrystalline TODGA-lanthanide complexes that are otherwise not prone to crystallization by use of OMC nanoparticles represents a new entry to addressing long-standing issues in purification by liquid-solid phase separation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2012

Nucleobases and other Prebiotic Species from the Ultraviolet Irradiation of Pyrimidine in Astrophysical Ices

Nucleobases are N-heterocycles that are the informational subunits of DNA and RNA, and are divided into two families: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites and their extraterrestrial origin confirmed by isotope measurement. Although no Nheterocycles have ever been observed in the ISM, the positions of the 6.2-m interstellar emission features suggest a population of such molecules is likely to be present. In this work we study the formation of pyrimidine-based molecules, including nucleobases, as well as other species of prebiotic interest, from the ultraviolet (UV) irradiation of pyrimidine in combinations of H2O, NH3, CH3OH, and CH4 ices at low temperature, in order to simulate the astrophysical conditions under which prebiotic species may be formed in the interstellar medium and icy bodies of the Solar System. Experimental: Gas mixtures are prepared in a glass mixing line (background pressure approx. 10(exp -6)-10(exp -5) mbar). Relative proportions between mixture components are determined by their partial pressures. Gas mixtures are then deposited on an aluminum foil attached to a cold finger (15-20 K) and simultaneously irradiated with an H2 lamp emitting UV photons (Lyman and a continuum at approx.160 nm). After irradiation samples are warmed to room temperature, at which time the remaining residues are recovered to be analyzed with liquid and gas chromatographies. Results: These experiments showed that the UV irradiation of pyrimidine mixed in these ices at low temperature leads to the formation of several photoproducts derived from pyrimidine, including the nucleobases uracil and cytosine, as well as their precursors 4(3H)-pyrimidone and 4-aminopyrimidine (Fig. 1). Theoretical quantum calculations on the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in pure H2O ices are in agreement with their experimental formation pathways. In those residues, other species of prebiotic interest such as urea and the amino acids glycine and alanine could also be identified. However, no pyrimidine derivatives containing CH3 groups, including the third nucleobase thymine, could be identified, suggesting that the addition of methyl groups to pyrimidine is not an efficient process.

DNA and RNA↗
DOE OSTI2025

Evaluating inkjet dispenser/liquid vortex capture-mass spectrometry for single-cell metabolomics in Hep G2 steatosis caused by tamoxifen

Single-cell mass spectrometry (MS) is advancing our understanding of metabolic pathways in heterogeneous cell populations; however, many techniques are slow or require disruptive sample preparations. This study evaluated coupling a modified HP D100 single-cell inkjet dispenser with liquid vortex capture-mass spectrometry (D100/LVC-MS). The D100 is a single-cell inkjet dispenser capable of titrating solutions and isolating single cells via disposable cassettes equipped with microfluidic channels and an impedance sensor. The LVC-MS enables high-throughput capture, lysis, and ionization of analytes for mass spectrometric analysis. The D100/LVC-MS system was characterized through titration and single-cell experiments. Propranolol titration demonstrated linearity across a broad concentration range using the D100/LVC-MS system. Additionally, Hep G2 hepatocarcinoma cells and Chlamydomonas reinhardtii algae were used to showcase the D100’s high-throughput or low-buffer-volume single-cell dispensing strategies. The D100/LVC-MS system’s performance was validated by evaluating tamoxifen-induced steatosis in Hep G2 cells. Tamoxifen, associated with nonalcoholic fatty liver disease in breast cancer patients following long-term use, was tested in Hep G2 cells at 20 µM for 72 h against DMSO-treated controls. High-throughput analysis of 500 cells per condition, completed in 25 min per run, demonstrated the system’s efficiency. The D100/LVC-MS system simultaneously quantified tamoxifen and measured triglycerides and phosphatidylcholines. Triglycerides were upregulated in the tamoxifen-treated cells and the results indicated two distinct cell populations, differing in tamoxifen and phosphatidylcholines levels, suggesting heterogeneity within the treated population. In conclusion, these findings highlight the D100/LVC-MS system as a cost-effective, high-throughput platform for single-cell metabolomics and lipidomics, with significant potential for evaluating metabolic alterations.

Chromatography↗
DOE OSTI2026

Temperature-dependent changes in gas chromatographic separation metrics for trihexyl(tetradecyl)phosphonium-based ionic liquid stationary phases and comparison to conventional polysiloxane stationary phases

Here, to assess whether the trihexyl(tetradecyl)phosphonium chloride ([P 66614 + ][Cl - ]) ionic liquid (IL), employed as a gas chromatographic stationary phase, exhibits temperature-dependent phase transitions or structural heterogeneity, retention thermodynamics for probe molecules of varied chemical structure were investigated by gas chromatography (GC) over the temperature range of 25 to 64 °C using van’t Hoff analysis. All van’t Hoff plots for n-alkanes (C8–C12) and other probe analytes were linear, indicating the absence of detectable phase-transition behavior. The [P 66614 + ][Cl - ] IL was further compared with the [P 66614 + ] bis[(trifluoromethyl)sulfonyl]imide ([P 66614 + ][NTf 2 - ]) IL and commercial polysiloxane stationary phases in terms of temperature-dependent resolution behavior and chromatographic selectivity over the temperature range of 28–52 °C. The resolution factors of analytes in mixtures obtained using gas chromatography–mass spectrometry (GC–MS) and the summation of resolution values decreased smoothly with temperature on both IL stationary phases and on the dimethylphenylcyano-substituted polymeric stationary phase (OV-1701), whereas the poly(50% diphenyl/50% dimethyl siloxane) stationary phase (SPB-50) exhibited irregularities in its temperature-dependent summation of resolution profile, showing a distinct maximum in the temperature range of 34–40 °C. Selectivity values for the IL stationary phases were largely determined by the anion, with the [P 66614 + ][Cl - ] IL providing enhanced separation between polar and aromatic analytes, while the [P 66614 + ][NTf 2 - ] IL favored dispersive and π-π interactions that resulted in a reversal of elution order for some aliphatic–aromatic and alkyne-containing analyte pairs. Capillary columns prepared using different surface activation methods and coated with identical [P 66614 + ][Cl - ] IL stationary phase showed indistinguishable temperature-dependent trends in summation of resolution value and selectivity. An evaluation of analyte-specific selectivity provides insight into the characteristic separation behavior of the stationary phases and enables identification of analyte classes that are more efficiently separated on the [P 66614 + ]-based IL stationary phases compared to conventional stationary phase materials.

Chromatographic selectivity↗
DOE OSTI2025

Crosslinked polymeric ionic liquid stationary phases incorporating a combination of silver(I) and copper(II) ions for olefin separations by gas chromatography

The development of efficient and selective olefin separation technologies is a critical challenge within the chemical industry given the high energy demands of conventional cryogenic distillation methods. To address this, the study explores the effectiveness of polymeric ionic liquid (PIL) stationary phases in gas chromatography (GC) that incorporate both silver(I) ([Ag + ]) and copper(II) ([Cu 2+ ]) ions for enhanced olefin separation via π-complexation interactions. Further, the effects of both non-crosslinked and crosslinked PIL stationary phases containing varying ratios of [Ag⁺] and [Cu²⁺] ions were systematically investigated to evaluate their impact on the separation of olefins from paraffins and structurally similar olefins. Retention measurements using a broad range of olefinic probes revealed that [Ag⁺] ions provided stronger and more consistent π-complexation in PIL-based stationary phases, while [Cu²⁺] ions preferentially enhance olefin retention in crosslinked systems. Furthermore, thermal stability studies showed that [Ag + ]-containing PIL stationary phases maintain stable olefin separation efficiency over prolonged exposure under elevated temperatures, while crosslinked PIL stationary phases containing [Cu 2+ ] ions exhibited stronger retention due to increased surface adsorption. These findings provide insight into the tunability of metal–olefin interactions using PIL-based stationary phases and suggest design strategies for achieving thermally stable and highly selective chromatographic separations of unsaturated hydrocarbons.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Polymeric ionic liquids containing copper(I) and copper(II) ions as gas chromatographic stationary phases for olefin separations

Copper(I) ions (Cu + ) are used in olefin separations due to their olefin complexing ability and low cost, but their instability in the presence of water and gases limits their widespread use. Ionic liquids (ILs) have emerged as stabilizers of Cu + ions and prevent their degradation, providing high olefin separation efficiency. There is limited understanding into the role that polymeric ionic liquids (PILs), which possess similar structural characteristics to ILs, have on Cu + ion-olefin interactions. Moreover, copper ions with diverse oxidation states, including Cu + and Cu 2+ ions, have been rarely employed for olefin separations. In this study, gas chromatography (GC) is used to investigate the interaction strength of olefins to stationary phases composed of the 1-hexyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([C 6 MIM + ][NTf 2 – ]) IL and the poly(1-hexyl-3-vinylimidazolium [NTf 2 – ]) (poly([C 6 VIM + ][NTf 2 – ])) PIL containing monovalent and divalent copper salts (i.e., [Cu + ][NTf 2 – ] and [Cu 2+ ]2[NTf 2 – ]). The chromatographic retention of alkenes, alkynes, dienes, and aromatic compounds was examined. Incorporation of the [Cu 2+ ]2[NTf 2 – ] salt into a stationary phase comprised of poly(dimethylsiloxane) resulted in strong retention of olefins, while its addition to the [C 6 MIM + ][NTf 2 – ] IL and poly([C 6 VIM + ][NTf 2 – ]) PIL allowed for the interaction strength to be modulated. Olefins exhibited greater affinities toward IL and PIL stationary phases containing the [Cu 2+ ]2[NTf 2 – ] salt compared to those with the [Cu + ][NTf 2 – ] salt. Elimination of water from both copper salts was observed to be an important factor in promoting olefin interactions, as evidenced by increased olefin retention upon exposure of the stationary phases to high temperatures. Furthermore, to evaluate the long-term thermal stability of the stationary phase, chromatographic retention of probes was measured on the [Cu 2+ ]2[NTf 2 – ]/[C 6 MIM + ][NTf 2 – ] IL stationary phase after its exposure to helium at a temperature of 110°C.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1992

Distribution of iodine into blood components of the Sprague-Dawley rat differs with the chemical form administered

It has been reported previously that radioactivity derived from iodine distributes differently in the Sprague-Dawley rat depending on the chemical form administered (Thrall and Bull, 1990). In the present communication we report the differential distribution of radioactivity derived from iodine (I2) and iodide (I-) into blood components. Twice as much radioiodine is in the form of I- in the plasma of animals treated with 125I- compared to 125I2-treated rats. No I2 could be detected in the plasma. With an increase in dose, increasing amounts of radioactivity derived from 125I2-treated animals distribute to whole blood compared to equivalent doses of 125I-, reaching a maxima at a dose of 15.8 mumol I/kg body weight. Most of the radioactivity derived from I2 associates with serum proteins and lipids, in particular with albumin and cholesteryl iodide. These data indicate a differential distribution of radioactivity depending on whether it is administered as iodide or iodine. This is inconsistent with the commonly held view that iodine (I2) is reduced to iodide (I-) before it is absorbed systemically from the gastrointestinal tract.

Non-NASA Center↗
DOE OSTI2020

Direct Observation of Liquid Crystal Droplet Configurational Transitions using Optical Tweezers

Liquid crystals (LCs) are easily influenced by external interactions, particularly at interfaces. When rod-like LC molecules are confined to spherical droplets, they experience a competition between interfacial tension and elastic deformations. The configuration of LCs inside a droplet can be controlled using surfactants that influence the interfacial orientation of the LC molecules in the oil-phase of an oil in water emulsion. Here, we used the surfactant sodium dodecyl sulfate (SDS) to manipulate the orientation of 5CB molecules in a polydisperse emulsion and examined the configuration of the droplets as a function of SDS concentration. We triggered pronounced morphological transitions by altering the SDS concentration while observing an individual LC droplet held in place using an optical tweezer. We compared the experimental configuration changes to predictions from simulations. Finally, we observed a hysteresis in the SDS concentration that induced the morphological transition from radial to bipolar and back as well as a fluctuations in the configuration during the transition.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Deep eutectic solvents as green and sustainable diluents in headspace gas chromatography for the determination of trace level genotoxic impurities in pharmaceuticals

Genotoxic impurities (GTIs) are potential carcinogens that need to be controlled down to ppm or lower concentration levels in pharmaceuticals under strict regulations. The static headspace gas chromatography (HS-GC) coupled with electron capture detection (ECD) is an effective approach to monitor halogenated and nitroaromatic genotoxins. Deep eutectic solvents (DESs) possess tunable physico-chemical properties and low vapor pressure for HS-GC methods. In this study, zwitterionic and non-ionic DESs have been used for the first time to develop and validate a sensitive analytical method for the analysis of 24 genotoxins at sub-ppm concentrations. Compared to non-ionic diluents, zwitterionic DESs produced exceptional analytical performance and the betaine: 7 (1,4- butane diol) DES outperformed the betaine: 5 (1,4-butane diol) DES. Limits of detection (LOD) down to the 5-ppb concentration level were achieved in DESs. Wide linear ranges spanning over 5 orders of magnitude (0.005–100 µg g –1 ) were obtained for most analytes with exceptional sensitivities and high precision. The method accuracy and precision were validated using 3 commercially available drug substances and excellent recoveries were obtained. Finally, this study broadens the applicability of HS-GC in the determination of less volatile GTIs by establishing DESs as viable diluent substitutes for organic solvents in routine pharmaceutical analysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Role of karyopherin nuclear transport receptors in nuclear transport by nuclear trafficking peptide

Nuclear trafficking peptide (NTP), a cell-penetrating peptide (CPP) composed of 10 amino acids (aa) (RIFIHFRIGC), has potent nuclear trafficking activity. Recently, we established a protein-based cell engineering system by using NTP, but it remained elusive how NTP functions as a CPP with nuclear orientation. In the present study, we identified importin subunit β1 (IMB1) and transportin 1 (TNPO1) as cellular proteins underlying the activity of NTP. These karyopherin nuclear transport receptors were identified as candidate molecules by liquid chromatography/mass spectrometry analysis, and downregulation of each protein by small interfering RNA significantly reduced NTP activity (P < 0.01). Biochemical analyses revealed that NTP bound directly to both molecules, and the forced expression of an IMB1 fragment (296–516 aa) or TNPO1 fragment (1–297 aa), which both contain binding sites to NTP, reduced nuclear NTP-green fluorescent protein (GFP) levels when it was added to cell culture medium. NTP is derived from viral protein R (Vpr) of human immunodeficiency virus-1, and Vpr enters the nucleus and exerts pleiotropic functions. Notably, Vpr bound directly to IMB1 and TNPO1, and its function was significantly impaired by the forced expression of the 296–516-aa fragment of IMB1 and 1–297-aa fragment of TNPO1. Interestingly, NTP completely blocked the physical association of Vpr with IMB1 and TNPO1. Although the nuclear localization mechanism of Vpr remains unknown, our data suggest that NTP functions as a novel nuclear localization signal of Vpr.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Proteomic Characterization of Damaged Single Hairs Recovered after an Explosion for Protein-Based Human Identification

Evidence recovery is challenging where an explosion has occurred. Though hair evidence may be sufficiently robust to be recovered at the site, forensic analysis underutilizes the matrix by relying on morphological analysis. Where DNA is compromised, particularly in hair, protein-based human identification presents a promising alternative. Detection of amino acid polymorphisms in hair proteins as genetically variant peptides (GVPs) permits the inference of individualizing single nucleotide polymorphisms for identification. However, an explosive blast may damage hair proteins and compromise GVP identification. This work evaluates effects of an explosive blast on the hair proteome and GVP identification, investigates microscopy as a predictor of proteome profiling success in recovered hairs to improve analysis throughput, and quantifies discriminative power in damaged hairs. The proteomics dataset has been deposited into the ProteomeXchange Consortium (PXD017427). With the exception of degradation in keratins K75, K80, K40, and keratin-associated protein KAP10-11 as markers of hair cuticular damage, corroborated by scanning electron microscopic analysis, minimal hair proteome degradation following explosion allowed successful proteome profiling of single hairs regardless of morphological damage. Finally, GVP identification remained independent of explosion conditions, permitting similar discriminative power between exploded and undamaged hairs. These findings lend greater confidence to GVP analysis in one-inch hairs for forensic identification and provide information about hair protein localization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

MARLOWE: An Untargeted Proteomics, Statistical Approach to Taxonomic Classification for Forensics

General proteomics research for fundamental science typically addresses laboratory- or patient-derived samples of known origin and composition. However, in a few research areas, such as environmental proteomics, clinical identification of infectious organisms, archeology, art/cultural history, and forensics, attributing the origin of a protein-containing sample to the organisms that produced it is a central focus. A small number of groups have approached this problem and developed software tools for taxonomic characterization and/or identification using bottom-up proteomics. Most such tools identify peptides via database search, and many rely on organism-specific peptides as markers. Our group recently introduced MARLOWE, a software tool for taxonomic characterization of unknown samples based on de novo peptide identification and signal-erosion-resistant strong peptides, which are shared peptides distributed in a taxonomy-dependent manner. In the current work, we further characterize the utility of MARLOWE using publicly available proteomics data from forensically-relevant samples. MARLOWE characterizes samples based on their protein profile, and returns ranked organism lists of potential contributors and taxonomic scores based on shared strong peptides between organisms. Overall, the correct characterization rate ranges between 44 and 100%, depending on the sample type and data acquisition parameters (with lower numbers associated with lower-quality data sets). MARLOWE demonstrates successful characterization of true contributors and close relatives, and provides sufficient specificity to distinguish certain microbial species. MARLOWE demonstrates its ability to provide insight into potential taxonomic sources for a wide range of sample types without prior assumptions about sample contents. As a result, this approach can find utility in forensic science and also broadly in bioanalytical applications that utilize proteomics approaches for taxonomic characterization.

Bacteria↗
DOE OSTI2020

Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. Furthermore, the methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Neurotransmitter networks in mouse prefrontal cortex are reconfigured by isoflurane anesthesia

This study quantified eight, small molecule neurotransmitters collected simultaneously from prefrontal cortex of C57BL/6J mouse (n=23) during wakefulness and during isoflurane anesthesia (1.3%). Using isoflurane anesthesia as an independent variable enabled evaluation of the hypothesis that isoflurane anesthesia differentially alters concentrations of multiple neurotransmitters and their interactions. Machine learning was applied to reveal higher order interactions among neurotransmitters. Using a between-subjects design, microdialysis was performed during wakefulness and during anesthesia. Concentrations (nM) of acetylcholine, adenosine, dopamine, GABA, glutamate, histamine, norepinephrine, and serotonin in the dialysis samples are reported (mean ± SD). Relative to wakefulness, acetylcholine concentration was lower during isoflurane anesthesia (1.254 ± 1.118 versus 0.401 ± 0.134, P=0.009), and concentrations of adenosine (29.456 ± 29.756 versus 101.321 ± 38.603, P<0.001), dopamine (0.0578 ± 0.0384 versus 0.113 ± 0.084, P=0.036), and norepinephrine (0.126 ± 0.080 versus 0.219 ± 0.066, P=0.010) were higher during anesthesia. Isoflurane reconfigured neurotransmitter interactions in prefrontal cortex, and the state of isoflurane anesthesia was reliably predicted by prefrontal cortex concentrations of adenosine, norepinephrine, and acetylcholine. A novel finding to emerge from machine learning analyses is that neurotransmitter concentration profiles in mouse prefrontal cortex undergo functional reconfiguration during isoflurane anesthesia. Adenosine, norepinephrine, and acetylcholine showed high feature importance, supporting the interpretation that interactions among these three transmitters may play a key role in modulating levels of cortical and behavioral arousal.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Proteomics Analysis of Human Contaminant Proteins

Complete characterization of unknowns via proteomics remains challenging. There exist regions of mass spectrometry-based proteomics data where empirical measurements are not attributed to peptides, and/or sequenced peptides from mass spectra are not attributed to any source. These uncharacterized regions are known as the “dark” proteome. Many proteomics tools rely on some a priori knowledge of sample composition; few tools allow for investigation of unknowns without relying on composition assumptions. Further, the potential low abundance of minor traces in these uncharacterized regions can make elucidation of the “dark” proteome challenging. Herein, we describe the development and evaluation of approaches to study the “dark” proteome and move towards an untargeted approach for more complete characterization, namely by studying minor human protein traces in non-human samples and combining that approach with non-human source organism identification without relying on assumptions. Human protein markers, in the form of genetically variant peptides, have been extensively examined in a variety of human matrices, including blood, plasma, and hair, but have yet to be investigated in non-human samples, such as cell cultures, as human contaminant traces. Genetically variant peptides are those that are found in proteins carrying single nucleotide polymorphisms. In this work, we aimed to (1) investigate the feasibility of detecting human contaminant genetically variant peptides (GVPs) in a diverse set of non-human organisms using public proteomics data and a computational pipeline, as well as to (2) develop a combined capability for untargeted source organism characterization and GVP detection. To our knowledge, this is the first report of applying these approaches towards a more complete proteomic characterization of unknowns. We successfully demonstrate the feasibility of broad human contaminant GVP detection in proteomics data, develop a better understanding of GVP detectability, characterize the sample-to-sample variability in GVP detection, and identify a core set of GVPs that can potentially be used as markers indicative of the human contaminant traces portion of the “dark” proteome. Further, we developed and evaluated a combined pipeline, MARLOWE-GVP, that enables both untargeted source organism characterization and GVP detection. We show high accuracy of correct source organism characterization and high degree of similarity of human contaminant GVP detection compared to the conventional approach. Success on both these efforts have allowed us to advance our understanding and characterization of the “dark” proteome.

59 BASIC BIOLOGICAL SCIENCES↗