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DOE OSTI2020

Materials Data on RbCS(OF)3 by Materials Project

RbCS(OF)3 crystallizes in the monoclinic P2_1 space group. The structure is two-dimensional and consists of one RbCS(OF)3 sheet oriented in the (0, 0, 1) direction. Rb1+ is bonded in a 8-coordinate geometry to seven O2- and one F1- atom. There are a spread of Rb–O bond distances ranging from 2.90–3.50 Å. The Rb–F bond length is 3.21 Å. C4+ is bonded in a trigonal non-coplanar geometry to three F1- atoms. There are a spread of C–F bond distances ranging from 1.35–1.37 Å. S4+ is bonded in a trigonal non-coplanar geometry to three O2- atoms. All S–O bond lengths are 1.46 Å. There are three inequivalent O2- sites. In the first O2- site, O2- is bonded in a distorted single-bond geometry to two equivalent Rb1+ and one S4+ atom. In the second O2- site, O2- is bonded in a distorted single-bond geometry to two equivalent Rb1+ and one S4+ atom. In the third O2- site, O2- is bonded in a single-bond geometry to three equivalent Rb1+ and one S4+ atom. There are three inequivalent F1- sites. In the first F1- site, F1- is bonded in a single-bond geometry to one C4+ atom. In the second F1- site, F1- is bonded in a single-bond geometry to one C4+ atom. In the third F1- site, F1- is bonded in a single-bond geometry to one Rb1+ and one C4+ atom.

36 MATERIALS SCIENCE↗

DOE OSTI2020

Materials Data on RbCS(OF)3 by Materials Project

RbCS(OF)3 crystallizes in the monoclinic Cm space group. The structure is three-dimensional. there are five inequivalent Rb1+ sites. In the first Rb1+ site, Rb1+ is bonded in a 8-coordinate geometry to nine O2- and one F1- atom. There are a spread of Rb–O bond distances ranging from 2.96–3.61 Å. The Rb–F bond length is 3.43 Å. In the second Rb1+ site, Rb1+ is bonded in a 7-coordinate geometry to six O2- and one F1- atom. There are a spread of Rb–O bond distances ranging from 2.90–3.28 Å. The Rb–F bond length is 3.34 Å. In the third Rb1+ site, Rb1+ is bonded in a 7-coordinate geometry to nine O2- and two equivalent F1- atoms. There are a spread of Rb–O bond distances ranging from 2.90–3.57 Å. Both Rb–F bond lengths are 3.60 Å. In the fourth Rb1+ site, Rb1+ is bonded in a 3-coordinate geometry to six O2- and three F1- atoms. There are a spread of Rb–O bond distances ranging from 2.74–3.40 Å. There are a spread of Rb–F bond distances ranging from 2.84–3.35 Å. In the fifth Rb1+ site, Rb1+ is bonded in a 11-coordinate geometry to nine O2- and two equivalent F1- atoms. There are a spread of Rb–O bond distances ranging from 3.07–3.64 Å. Both Rb–F bond lengths are 3.46 Å. There are five inequivalent C4+ sites. In the first C4+ site, C4+ is bonded in a trigonal non-coplanar geometry to three F1- atoms. There are a spread of C–F bond distances ranging from 1.35–1.37 Å. In the second C4+ site, C4+ is bonded in a trigonal non-coplanar geometry to three F1- atoms. There are a spread of C–F bond distances ranging from 1.35–1.37 Å. In the third C4+ site, C4+ is bonded in a trigonal non-coplanar geometry to three F1- atoms. All C–F bond lengths are 1.36 Å. In the fourth C4+ site, C4+ is bonded in a trigonal non-coplanar geometry to three F1- atoms. There is one shorter (1.35 Å) and two longer (1.36 Å) C–F bond length. In the fifth C4+ site, C4+ is bonded in a trigonal non-coplanar geometry to three F1- atoms. There is one shorter (1.35 Å) and two longer (1.36 Å) C–F bond length. There are five inequivalent S4+ sites. In the first S4+ site, S4+ is bonded in a trigonal non-coplanar geometry to three O2- atoms. There is two shorter (1.46 Å) and one longer (1.47 Å) S–O bond length. In the second S4+ site, S4+ is bonded in a trigonal non-coplanar geometry to three O2- atoms. There is two shorter (1.46 Å) and one longer (1.47 Å) S–O bond length. In the third S4+ site, S4+ is bonded in a trigonal non-coplanar geometry to three O2- atoms. All S–O bond lengths are 1.46 Å. In the fourth S4+ site, S4+ is bonded in a trigonal non-coplanar geometry to three O2- atoms. There is two shorter (1.46 Å) and one longer (1.47 Å) S–O bond length. In the fifth S4+ site, S4+ is bonded in a trigonal non-coplanar geometry to three O2- atoms. All S–O bond lengths are 1.46 Å. There are thirteen inequivalent O2- sites. In the first O2- site, O2- is bonded in a distorted single-bond geometry to two equivalent Rb1+ and one S4+ atom. In the second O2- site, O2- is bonded in a single-bond geometry to three Rb1+ and one S4+ atom. In the third O2- site, O2- is bonded in a distorted single-bond geometry to three Rb1+ and one S4+ atom. In the fourth O2- site, O2- is bonded in a single-bond geometry to three Rb1+ and one S4+ atom. In the fifth O2- site, O2- is bonded in a distorted single-bond geometry to two Rb1+ and one S4+ atom. In the sixth O2- site, O2- is bonded in a single-bond geometry to three Rb1+ and one S4+ atom. In the seventh O2- site, O2- is bonded in a distorted single-bond geometry to two Rb1+ and one S4+ atom. In the eighth O2- site, O2- is bonded in a single-bond geometry to two equivalent Rb1+ and one S4+ atom. In the ninth O2- site, O2- is bonded in a single-bond geometry to three Rb1+ and one S4+ atom. In the tenth O2- site, O2- is bonded in a distorted single-bond geometry to two Rb1+ and one S4+ atom. In the eleventh O2- site, O2- is bonded in a distorted single-bond geometry to three Rb1+ and one S4+ atom. In the twelfth O2- site, O2- is bonded in a distorted single-bond geometry to two Rb1+ and one S4+ atom. In the thirteenth O2- site, O2- is bonded in a distorted single-bond geometry to three Rb1+ and one S4+ atom. There are thirteen inequivalent F1- sites. In the first F1- site, F1- is bonded in a single-bond geometry to one C4+ atom. In the second F1- site, F1- is bonded in a single-bond geometry to one C4+ atom. In the third F1- site, F1- is bonded in a single-bond geometry to one Rb1+ and one C4+ atom. In the fourth F1- site, F1- is bonded in a single-bond geometry to one Rb1+ and one C4+ atom. In the fifth F1- site, F1- is bonded in a single-bond geometry to one Rb1+ and one C4+ atom. In the sixth F1- site, F1- is bonded in a single-bond geometry to one C4+ atom. In the seventh F1- site, F1- is bonded in a single-bond geometry to one Rb1+ and one C4+ atom. In the eighth F1- site, F1- is bonded in a single-bond geometry to one Rb1+ and one C4+ atom. In the ninth F1- site, F1- is bonded in a single-bond geometry to one Rb1+ and one C4+ atom. In the tenth F1- site, F1- is bonded in a single-bond geometry to one Rb1+ and one C4+ atom. In the eleventh F1- site, F1- is bonded in a single-bond geometry to one C4+ atom. In the twelfth F1- site, F1- is bonded in a single-bond geometry to one C4+ atom. In the thirteenth F1- site, F1- is bonded in a single-bond geometry to one C4+ atom.

36 MATERIALS SCIENCE↗

NASA NTRSDate not supplied

Mechanical Unloading Effects Hematopoiesis and Subsequent Red Blood Cell Life Cycle Evaluated at Singe Cell Resolution

Red blood cell formation or erythropoiesis in the bone marrow and exit into circulation are known to be affected by mechanical loading. Specifically, space anemia is a condition observed during prolonged microgravity unloading in which the red blood cell (RBC) mass in circulation is reduced. However, the mechanisms causing space anemia are yet to be clearly established, but may include increased hemolysis of RBC in circulation, decreased RBC production or a combination of both factors. The presentation of space anemia is comparable to chronic anemia conditions associated with aging, disuse, and poor health. Molecular dysregulation in the RBC's life cycle is poorly defined and is a significant concern in the context of disuse anemia as mechanotransduction significantly alters of gene expression, protein synthesis and cell signaling pathways. Our study analyzes mouse single cell RNA sequencing libraries from the bone marrow, circulating blood, and spleen and integrates the RBC lineage populations from the tissues into a RBC life cycle atlas. This study also probes how age at time of spaceflight contributes to the molecular signature of space anemia. The overarching objective of the study is to develop a mechanistic understanding of space anemia and translate the findings to a better understanding of aging, disuse, and poor health-related anemia to defining the role of mechanical loading in health and devise potential therapeutic interventions. Important findings of our study are: 1) that mechanical unloading in microgravity alters the proportion of developmental RBC lineage cells, skewing toward progenitor, indicating that fewer RBCs may be present in blood; 2) that late reticulocyte number is elevated in the bone marrow of older animals, suggesting a failure to enter circulation; and 3) that the molecular signature of the circulating RBCs is not significantly altered in microgravity. However, expression markers for eryptosis and erythrophagocytosis are elevated in circulating and spleen RBCs, suggesting spaceflight may shorten the lifetime of RBCs after exit from the marrow. In conclusion our results suggest early stages of erythropoiesis proliferation and differentiation in the marrow are key factors in space anemia and mechanical loading may be a necessary step of RBC entry in circulation.

blood↗

DOE OSTI2026

Microphase Separation of Randomly Linked Branched Polystyrene/Polylactic Acid for Formation of Cocontinuous Nanostructures

Cocontinuous polymeric nanomaterials have gained attention for their ability to preserve distinct properties of constituent microphases within a single material. Randomly linked copolymer networks have shown very wide stability windows for disordered cocontinuous phases (extending over ≈ 30 wt % in composition), but the reliance on a network architecture prevents subsequent solution- or melt-processing. Furthermore, the key factors contributing to cocontinuity have remained unclear. We recently found that randomly linked star copolymers (RSCs) can exhibit a cocontinuous window as wide as 25 wt % in the case of 4-arm stars, suggesting that while a network architecture is not essential for the formation of disordered cocontinuous phases, the presence of random elastic forces in such architectures may indeed facilitate their formation. In addition, the behavior was found to be highly sensitive to arm number, with 6-arm RSCs exhibiting almost no cocontinuous phase. These results raised a key mechanistic question regarding the contribution of random elastic forces, originating from strands that bridge between junctions, in stabilizing disordered cocontinuous phases. In the current study, we synthesized randomly linked branched copolymers (RBCs) of polystyrene (PS) and poly(D,L-lactic acid) (PLA), which represent an intermediate architecture between networks and stars. This approach allows for the introduction of elastic contributions from strands bridging between different junctions, while still maintaining the processability advantages of a non-network architecture. The cocontinuous regions of the PS/PLA RBCs, with varying polymer and linker functionalities (f p and f l , respectively), were characterized by small-angle X-ray scattering, gravimetry, and scanning electron microscopy. We found that the cocontinuous windows of RBCs typically expanded with increasing elastic contributions and exhibited reduced sensitivity to junction-functionality compared to RSCs. Notably, RBCs with f p = 1.50 and f l = 3, which had large molecular weights due to proximity to the gel point, achieved a cocontinuous window of ≈ 34 wt %, which is almost twice as wide as analogous 3-arm RSCs and comparable to randomly linked networks. Leveraging this robust cocontinuity and solution-processability, we fabricated a film of interconnected nanoporous PS.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS1971

Anaerobic glycolysis and specific gravity of the red blood cells of rats exposed to pure oxygen at 600 torr.

Rats were exposed to 100% oxygen at 600 torr for up to 8 days. Highly significant increases in RBC anaerobic glycolysis occurred during the first 4 days of exposure and then subsided. Two significant peaks were found, one on days 1 and 2 and one on day 4. The first peak is attributed to reticulocytosis, which was maximal after 90 minutes and had disappeared by day 3. A second mechanism must account for the peak on day 4. An interpretation of the second peak is provided by existing evidence that selective removal of older RBCs occurs during the first few days of exposure to hypobaric oxygen, with maximum effect on day 4. Results in splenectomized, sham-operated and intact animals were indistinguishable from each other. A significant decrease in RBC specific gravity was found in exposed animals with spleens intact, but not in splenectomized animals. Theoretical aspects of age-related parameters as an aid to continuous detection and evaluation of changes in RBC populations are discussed.

Sabine, J. C.↗

DOE OSTI2023

Sample Preparation Methods for Targeted Single-Cell Proteomics

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540–660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390–540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5–25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate

Background: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. Methods: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. Results: Purified nHRP2 was identified by SDS-PAGE and western blot as a –60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. Conclusions: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.

59 BASIC BIOLOGICAL SCIENCES↗