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Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been engineered to replace or augment the oxygen carrying capacity of erythrocytes. However, clinical results have generally been disappointing, in part due to the intrinsic oxidative toxicity of Hb. The most common HBOC starting material is adult human or bovine Hb. However, it has been suggested that fetal Hb may offer advantages due to decreased oxidative reactivity. Large-scale manufacturing of HBOC will likely and ultimately require recombinant sources of human proteins. We, therefore, directly compared the functional properties and oxidative reactivity of recombinant fetal (rHbF) and recombinant adult (rHbA) Hb. rHbA and rHbF produced similar yields of purified functional protein. No differences were seen in the two proteins in: autoxidation rate; the rate of hydrogen peroxide reaction; NO scavenging dioxygenase activity; and the NO producing nitrite reductase activity. The rHbF protein was: less damaged by low levels of hydrogen peroxide; less damaging when added to human umbilical vein endothelial cells (HUVEC) in the ferric form; and had a slower rate of intrinsic heme loss. The rHbA protein was: more readily reducible by plasma antioxidants such as ascorbate in both the reactive ferryl and ferric states; less readily damaged by lipid peroxides; and less damaging to phosphatidylcholine liposomes. In conclusion in terms of oxidative reactivity, there are advantages and disadvantages to the use of rHbA or rHbF as the basis for an effective HBOC.

Platelet extracellular vesicles (PEVs) have emerged as potential mediators in intercellular communication. PEVs exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. Here, imaging and analytical techniques were employed to unveil morphological pathways of the release, structure, composition, and surface properties of PEVs derived from human platelets (PLTs) activated with the thrombin receptor activating peptide (TRAP). Based on extensive electron microscopy analysis, we propose four morphological pathways for PEVs release from TRAP-activated PLTs: (1) plasma membrane budding, (2) extrusion of multivesicular α-granules and cytoplasmic vacuoles, (3) plasma membrane blistering and (4) “pearling” of PLT pseudopodia. The PLT extracellular vesiculome encompasses ectosomes, exosomes, free mitochondria, mitochondria-containing vesicles, “podiasomes” and PLT “ghosts”. Interestingly, a flow cytometry showed a population of TOM20 + LC3 + PEVs, likely products of platelet mitophagy. We found that lipidomic and proteomic profiles were different between the small PEV (S-PEVs; mean diameter 103 nm) and the large vesicle (L-PEVs; mean diameter 350 nm) fractions separated by differential centrifugation. In addition, the majority of PEVs released by activated PLTs was composed of S-PEVs which have markedly higher thrombin generation activity per unit of PEV surface area compared to L-PEVs, and contribute approximately 60% of the PLT vesiculome procoagulant potency.

We report an automated method for determining charge states from high-resolution mass spectra. Fourier transforms of isotope packets from high-resolution mass spectra are compared to Fourier transforms of modeled isotopic peak packets for a range of charge states. The charge state for the experimental ion packet is determined by the model isotope packet that yields the best match in the comparison of the Fourier transforms. This strategy is demonstrated for determining peptide ion charge states from \"zoom scan\" data from a linear quadrupole ion trap mass spectrometer, enabling the subsequent automated identification of singly- through quadruply-charged peptide ions, while reducing the numbers of conflicting identifications from ambiguous charge state assignments. We also apply this technique to determine the charges of intact protein ions from LC-FTICR data, demonstrating that it is more sensitive under these experimental conditions than two existing algorithms. The strategy outlined in this paper should be generally applicable to mass spectra obtained from any instrument capable of isotopic resolution.

Background: Platelet (PLT) extracellular vesicles (PEVs) exhibit several activities with pathophysiological importance and may serve as diagnostic biomarkers. PLTs contain active autophagy/mitophagy pathways, however, association of PEV release with these processes have not been elucidated. Methods: The release of mitochondria (MITO) and autophagy associated PEVs from human PLTs activated by thrombin receptoractivating peptide (TRAP) was investigated by flow cytometry (FC), MS-proteomics, transmission electron microscopy (TEM), LASER-scanning confocal microscopy (LSCM) and nanoparticle tracking analysis. Results: Despite marked differences in proteomic profiles of large/dense PEVs (L-PeVs, obtained by 20,000 g spin; mean diameter 350 nm) and small PEVs (S-PEVs, obtained by 100,000 g spin of 20,000 g sup.; mean diameter 103 nm), both L-PEVs an S-PEVs carried proteasome complex and autophagy related proteins. MITO proteins including superoxide dismutase and ATP synthase were identified in both PEV fractions, though with lower abundance in the S-PEVs. TEM of PEVs showed damaged MITO, MITO fusing with cytoplasmic vacuoles and MITOcontaining vesicles. About 50% of FC-detectable PEVs (DHPE labeled) exposed the MITO marker TOM20; 16±1% of PEVs expressed the autophagosomal protein LC3 (LC3+ PEVs) and 12±3% of PEVs (75% of LC3+ PEVs) were TOM20+ LC3+ PEVs. Summary/Conclusion: Our results suggest that a portion of TOM20+ PEVs are mitophagosomes - products of PLT mitophagy. The formation of PEVs decorated with LC3 is indicative of a type-2 mitophagy in which LC3 bearing vesicles attach to damaged MITO, explaining the observed population of TOM20+ LC3+ PEVs. PLT mitophagosomes, their structure, mechanism of release and potential biologic effects need further investigation.

We present DARKNESS (the DARK-speckle Near-infrared Energy-resolving Superconducting Spectrophotometer), the first of several planned integral field spectrographs to use optical/near-infrared Microwave Kinetic Inductance Detectors (MKIDs) for high-contrast imaging. The photon counting and simultaneous low-resolution spectroscopy provided by MKIDs will enable real-time speckle control techniques and post-processing speckle suppression at frame rates capable of resolving the atmospheric speckles that currently limit high-contrast imaging from the ground. DARKNESS is now operational behind the PALM-3000 extreme adaptive optics system and the Stellar Double Coronagraph at Palomar Observatory. Here, we describe the motivation, design, and characterization of the instrument, early on-sky results, and future prospects.

Background LRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinson’s disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. Methods Capitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. Results pT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil degranulation, antigenic responses, and suppressed platelet activation. Conclusions The extracellular serum ratio of pT73-Rab10 to total Rab10 is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics that mitigate associated deleterious immunological responses.

Trihalomethanes (THMs) and haloacetic acids (HAAs) are regulated disinfection by-products (DBPs); their joint reproductive toxicity in drinking water is unknown. We aimed to evaluate a drinking water mixture of the four regulated THMs and five regulated HAAs in a multigenerational reproductive toxicity bioassay. Sprague-Dawley rats were exposed (parental, F1, and F2 generations) from gestation day 0 of the parental generation to postnatal day (PND) 6 of the F2 generation to a realistically proportioned mixture of THMs and HAAs at 0, 500×, 1,000×, or 2,000× of the U.S. Environmental Protection Agency's maximum contaminant levels (MCLs). Maternal water consumption was reduced at ≥ 1,000×; body weights were reduced at 2,000×. Prenatal and postnatal survival were unaffected. F1 pup weights were unaffected at birth but reduced at 2,000× on PND6 and at ≥ 1,000× on PND21. Postweaning F1 body weights were reduced at 2,000×, and water consumption was reduced at ≥ 500×. Males at 2,000× had a small but significantly increased incidence of retained nipples and compromised sperm motility. Onset of puberty was delayed at 1,000× and 2,000×. F1 estrous cycles and fertility were unaffected, and F2 litters showed no effects on pup weight or survival. Histologically, P0 (parental) dams had nephropathy and adrenal cortical pathology at 2,000×. A mixture of regulated DBPs at up to 2,000× the MCLs had no adverse effects on fertility, pregnancy maintenance, prenatal survival, postnatal survival, or birth weights. Delayed puberty at ≥ 1,000× may have been secondary to reduced water consumption. Male nipple retention and compromised sperm motility at 2,000× may have been secondary to reduced body weights.

We present the first results from the Revealing Low-Luminosity Active Galactic Nuclei (ReveaLLAGN) survey, a JWST survey of seven nearby LLAGNs. We focus on two observations with the Mid-Infrared Instrument (MIRI)’s Medium-Resolution Spectrometer of the nuclei of NGC 1052 and Sombrero (NGC 4594/M104). We also compare these data to public JWST data of higher-luminosity AGNs, NGC 7319 and NGC 7469. JWST clearly separates the AGN spectrum from the galaxy light even in Sombrero, the faintest target in our survey; the AGN components have very red spectra. We find that the emission-line widths in both NGC 1052 and Sombrero increase with increasing ionization potential, with FWHM > 1000 km s−1 for lines with ionization potential ≳ 50 eV. These lines are also significantly blueshifted in both LLAGNs. The high-ionization-potential lines in NGC 7319 show neither broad widths nor significant blueshifts. Many of the lower-ionization-potential emission lines in Sombrero show significant blue wings extending >1000 km s−1. These features and the emission-line maps in both galaxies are consistent with outflows along the jet direction. Sombrero has the lowest-luminosity high-ionization-potential lines ([Ne v] and [O iv]) ever measured in the mid-infrared, but the relative strengths of these lines are consistent with higher-luminosity AGNs. On the other hand, the [Ne v] emission is much weaker relative to the [Ne iii] and [Ne ii] lines of higher-luminosity AGNs. These initial results show the great promise that JWST holds for identifying and studying the physical nature of LLAGNs.

We present deep optical observations of the stellar halo of NGC 300, an LMC-mass galaxy, acquired with the DEEP subcomponent of the DECam Local Volume Exploration survey using the 4 m Blanco Telescope. Our resolved star analysis reveals a large, low surface brightness stellar stream (MV ∼ −8.5; [Fe/H] = −1.4 ± 0.15) extending more than 40 kpc north from the galaxy’s center. We also find other halo structures, including potentially an additional stream wrap to the south, which may be associated with the main stream. The morphology and derived low metallicities of the streams and shells discovered surrounding NGC 300 are highly suggestive of a past accretion event. Assuming a single progenitor, the accreted system is approximately Fornax-like in luminosity, with an inferred mass ratio to NGC 300 of approximately 1:15. We also present the discovery of a metal-poor globular cluster (GC) (Rproj = 23.3 kpc; MV = −8.99 ± 0.16; [Fe/H] ≈ −1.6 ± 0.6) in the halo of NGC 300, the furthest identified GC associated with NGC 300. The stellar structures around NGC 300 represent the richest features observed in a Magellanic Cloud analog to date, strongly supporting the idea that accretion and subsequent disruption is an important mechanism in the assembly of dwarf galaxy stellar halos.

Southeastern U.S. mobile and manufactured housing (MH) residents are the most tornado-vulnerable subset of the population because of both physical and socioeconomic factors. This study builds upon prior MH resident tornado vulnerability research by statistically and geographically analyzing responses from a survey administered to these residents in the Southeast. Specifically, 257 Alabama and Mississippi MH residents were administered a survey with questions pertaining to their perceived tornado risk and vulnerability, protective action and decision-making, and beliefs about the structural integrity of their homes. Results indicate that, despite the weather and emergency management enterprises consistently suggesting that MH residents evacuate their homes for sturdier shelter during tornado events, more than 50% of MH residents believe their homes are safe sheltering locations. The prevalence of larger MHs in northern Alabama partially influences willingness to shelter within one’s MH, while higher levels of negative affectivity stemming from recent impactful tornadoes in northern Alabama influences people to evacuate their MHs for safety. Study findings also uncovered a perception and vulnerability paradox for these residents: Those who have the means to evacuate their MH often feel they have no need to do so, whereas those who recognize the potential peril of sheltering in their home and want to evacuate often lack the resources and/or self-efficacy to carry out more desirable sheltering plans. Overall, study results provide valuable information for National Weather Service forecasters, emergency managers, and media partners so that they may use it for public outreach and MH resident education.