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At least 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Structural analysis of platelet fragments and extracellular vesicles produced by apheresis platelets during storage

Platelets (PLTs) for transfusion can be stored for up to 7 days at room temperature (RT). The quality of apheresis PLTs decreases over storage time, which affects PLT hemostatic functions. Here, we characterized the membranous particles produced by PLT storage lesion (PSLPs), including degranulated PLTs, PLT ghosts, membrane fragments, and extracellular membrane vesicles (PEVs). The PSLPs generated in apheresis platelet units were analyzed on days 1, 3, 5, and 7 of RT storage. A differential centrifugation and a sucrose density gradient were used to separate PSLP populations. PSLPs were characterized using scanning and transmission electron microscopy (EM), flow cytometry (FC), and nanoparticle tracking analysis (NTA). PSLPs have different morphologies and a broad size distribution; FC and NTA showed that the concentration of small and large PSLPs increases with storage time. The density gradient separated 3 PSLP populations: (1) degranulated PLTs, PLT ghosts, and large PLT fragments; (2) PEVs originated from PLT activation and organelles released by necrotic PLTs; and (3) PEV ghosts. Most PSLPs expressed phosphatidyl serine and induced thrombin generation in the plasma. PSLPs contained extracellular mitochondria and some had the autophagosome marker LC3. PSLPs encompass degranulated PLTs, PLT ghosts, large PLT fragments, large and dense PEVs, and low-density PEV ghosts. The activation-related PSLPs are released, particularly during early stage of storage (days 1-3), and the release of apoptosis- and necrosis-related PSLPs prevails after that. No elevation of LC3- and TOM20-positive PSLPs indicates that the increase of extracellular mitochondria during later-stage storage is not associated with PLT mitophagy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions

Here, bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host–pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host–pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by Salmonella enterica serovar Typhimurium ( S . Typhimurium) cultured under different conditions, which mimic host–pathogen interactions. S . Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium (phosphate-carbon-nitrogen, PCN), as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of Salmonella pathogenicity island 2 (SPI-2) genes. Moreover, Salmonella pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing S. Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts ( IsrB-1/2; IsrA; ffs; SsrS; CsrC; pSLT035; 10Sa; rnpB; STM0277; sseB; STM0972; STM2606 ), whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts ( SsrS, CsrC,10Sa, and rnpB ). In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1991

Critical behavior of two-dimensional vesicles in the deflated regime

The critical behavior of two-dimensional vesicles in the deflated regime is studied analytically using a mapping onto a gauge model, scaling arguments, and exact inequalities. In agreement with the results of earlier studies the critical behavior is governed by a branched-polymer fixed point. The shape of the critical line in the gauge model is deduced in the weak and in the infinitely deflated regime.

Banavar, Jayanth R.↗
NASA NTRS1995

Superradiance of J-Aggregated 2,2'-Cyanine Absorbed onto a Vesicle Surface

Phospholipid vesicles are used as substrates to form adsorbed aggregates of 2,2'-cyanine, also referred to as pseudoisocyanine (PIC). In this paper, we report photophysical parameters of two putative adsorbed aggregates species (cis- and trans-aggregates, relating to their makeup from mono-cis and all-transstereoisomers, respectively). Phase modulation picosecond fluorescence decay measurements reveal that superradiance and energy transfer are dominant features controlling photophysical processes. Superradiance, coherence size, energy transfer and exciton-phonon coupling are discussed for the two types of aggregates; as regards photophysical parameters, the fluorescence lifetimes, fluorescence quantum yields, and nonradiative rate constants are determined. It is suggested that structure plays the crucial role in excited state dynamics.

Akins, Daniel L.↗
NASA NTRS1996

Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

Faguy, David↗
DOE OSTI2026

Engineered Membrane Vesicle Production via oprF or oprI Deletion Has Distinct Phenotypic Effects in Pseudomonas putida -putative knockouts table

Table S1, putative gene knockout targets in P. putida KT2440 to enhance vesiculation; Table S2, protein sequence identity of OmpA from E. coli K12 to P. putida KT2440 genes; Table S3, strains utilized in this study and corresponding construction details; Table S4, oligonucleotides utilized in this study; Table S5, plasmids utilized in this study; Table S6, sequences for mNeonGreen, tags, and codon-optimized genes; Figure S1, particle count per gCDW for KT2440 and knockout strains corresponding to data presented in Figure 1B; Figure S2, OD600 measurements of extracted MVs from KT2440 and knockout strains; Figure S3, particle count per gCDW for WT, ΔPP_4669, and ΔPP_1502; Figure S4, particle count per gCDW for KT2440 and knockout strains corresponding to data presented in Figure 3C; Figure S5, sizes of MVs corresponding to particle counts in Figure S4; Figure S6, particle count per gCDW for KT2440 grown on 20 mM glucose alone or 20 mM glucose plus 12.5 mM p-coumarate and 12.5 mM ferulate; Figure S7 and Figure S8, principal component analysis of the cellular fractions; Figure S9, heatmap of outer membrane proteins with differential abundance; and Figure S10, mNeonGreen (mNG) fluorescence signal for the cellular fraction and the extracellular fraction

hypervesiculation↗
DOE OSTI2020

Hybrid Unilamellar Vesicles of Phospholipids and Block Copolymers with Crystalline Domains

Phospholipid (PL) membranes are ubiquitous in nature and their phase behavior has been extensively studied. Lipids assemble in a variety of structures and external stimuli can activate a quick switch between them. Amphiphilic block copolymers (BCPs) can self-organize in analogous structures but are mechanically more robust and transformations are considerably slower. The combination of PL dynamical behavior with BCP chemical richness could lead to new materials for applications in bioinspired separation membranes and drug delivery. It is timely to underpin the phase behavior of these hybrid systems and a few recent studies have revealed that PL–BCP membranes display synergistic structural, phase-separation, and dynamical properties not seen in pure components. One example is phase-separation in the membrane plane, which seems to be strongly affected by the ability of the PL to form lamellar phases with ordered alkyl chains. In this paper we focus on a rather less explored design handle which is the crystalline properties of the BCP component. Using a combination of confocal laser scanning microscopy and X-ray scattering we show that hybrid membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and methoxy-poly(ethylene glycol)-b-poly(ε-caprolactone) (mPEG-b-PCL) display BCP-rich and PL-rich domains when the BCP comprises crystalline moieties. The packing of the hydrophilic part of the BCP (PEG) favors mixing of DPPC at the molecular level or into nanoscale domains while semi-crystalline and hydrophobic PCL moieties bolster microscopic domain formation in the hybrid membrane plane

phase-separation↗
DOE OSTI2020

The C‐Terminal Domain of α‐Synuclein Confers Steric Stabilization on Synaptic Vesicle‐Like Surfaces

Abstract While α‐synuclein, an intrinsically disordered protein linked to Parkinson's disease, has been shown to associate with membrane organelles, its overall cellular function remains nebulous. α‐Synuclein binds to membranes through its amino‐terminal domain (first ≈100 residues), but there is no consensus on the biophysical function of the carboxyl‐terminal domain (last ≈40 residues) due, in part, to its lack of strong interaction partners and persisting intrinsic disorder even when membrane bound. Here, by directly applying force on α‐synuclein bound to spherical nanoparticle‐supported lipid bilayers (SSLBs) and tracking higher‐order structural changes through small‐angle X‐ray scattering, strong evidence is presented that α‐synuclein sterically stabilizes membrane surfaces through its carboxyl‐terminal domain. Full‐length α‐synuclein dramatically increases the critical osmotic pressure at which SSLBs cluster ( P C ≈ 1.3 × 10 5 Pa) compared to α‐synuclein without the carboxyl‐terminal domain ( P C ≈ 1.9 × 10 4 Pa) at physiological salt and temperature conditions. This clustering of α‐synuclein‐bound SSLBs is shown to be reversible and sensitive to monovalent/divalent salt, both features of grafted polyelectrolyte‐mediated steric stabilization. In elucidating the biophysical function of α‐synuclein in the framework of polymer science, it is demonstrated that the carboxyl‐terminal domain can potentially utilize its persisting intrinsic disorder to functionalize membrane surfaces.

Chung, Peter J.↗
DOE OSTI2019

Substrate-induced luminescence response of terbium-containing polymeric vesicles doped with the tetra- and bis-1,3-diketone derivatives of calix[4]arenes bearing nonyl substituents at the lower rims

Polymeric aggregates (~100 nm) based on polydiacetylenic acid doped with the luminescent Tb{sup III} complexes with the tetra- and bis-1,3-diketonate derivatives of calix[4]arenes with nonyl substituents at the lower rims are considered as potential nanosensors. The obtained colloidally stable mixed aggregates can generate a substrate-induced luminescent response in aqueous solutions of 2,2´,6´,2´´-terpyridine in the micromolar concentration range due to the coordination of the latter by Tb{sup III} ions on the aggregate surface.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗