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Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G ₁₂/₁₃ versus G q coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating G q-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G ₁₂/₁₃-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to G ᵢ and β-arrestin-1. Consistent with preferential G ₁₂/₁₃ coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with G q signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G ₁₂/₁₃ versus G q and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.

The unfavorable I–V characteristics of perovskite solar cells (PSCs), such as the I–V hysteresis phenomena, have been one major obstacle for their future practical application. However, corresponding analysis based on traditional theories have shown non-negligible flaws and failed for satisfactory explanation. To present a novel mechanism, here we utilize for the first time the memristive property of the perovskite material to analyze the I–V characteristics of PSCs. The obtained joint physical model and the deduced equation may help solving the long-existent mysteries of the I–V characteristics of PSCs. On the basis of our analysis and memristor theory, we also propose an original device optimization strategy for PSCs, which may help further increase their performance to the limit.

Dual-energy CT (DECT) with scans over limited-angular ranges (LARs) may allow reductions in scan time and radiation dose and avoidance of possible collision between the moving parts of a scanner and the imaged object. The beam-hardening (BH) and LAR effects are two sources of image artifacts in DECT with LAR data. In this work, we investigate a two-step method to correct for both BH and LAR artifacts in order to yield accurate image reconstruction in DECT with LAR data. From low- and high-kVp LAR data in DECT, we first use a data-domain decomposition (DDD) algorithm to obtain LAR basis data with the non-linear BH effect corrected for. We then develop and tailor a directional-total-variation (DTV) algorithm to reconstruct from the LAR basis data obtained basis images with the LAR effect compensated for. Finally, using the basis images reconstructed, we create virtual monochromatic images (VMIs), and estimate physical quantities such as iodine concentrations and effective atomic numbers within the object imaged. We conduct numerical studies using two digital phantoms of different complexity levels and types of structures. LAR data of low- and high-kVp are generated from the phantoms over both single-arc (SA) and two-orthogonal-arc (TOA) LARs ranging from 14∘ to 180∘. Visual inspection and quantitative assessment of VMIs obtained reveal that the two-step method proposed can yield VMIs in which both BH and LAR artifacts are reduced, and estimation accuracy of physical quantities is improved. In addition, concerning SA and TOA scans with the same total LAR, the latter is shown to yield more accurate images and physical quantity estimations than the former. We investigate a two-step method that combines the DDD and DTV algorithms to correct for both BH and LAR artifacts in image reconstruction, yielding accurate VMIs and estimations of physical quantities, from low- and high-kVp LAR data in DECT. The results and knowledge acquired in the work on accurate image reconstruction in LAR DECT may give rise to further understanding and insights into the practical design of LAR scan configurations and reconstruction procedures for DECT applications.

Prions are self-perpetuating and, in most cases, aggregation-prone protein isoforms that transmit neurodegenerative diseases in mammals and control heritable traits in yeast. Prion conversion requires a very high level of identity of the interacting protein sequences. Decreased transmission of the prion state between divergent proteins is termed \"species barrier\" and was thought to occur because of the inability of divergent prion proteins to coaggregate. Species barrier can be overcome in cross-species infections, e.g., from \"mad cows\" to humans. We studied the counterparts of yeast prion protein Sup35, originated from three different species of the Saccharomyces sensu stricto group and exhibiting the range of prion domain divergence that overlaps with the range of divergence observed among distant mammalian species. All three proteins were capable of forming a prion in Saccharomyces cerevisiae, although prions formed by heterologous proteins were usually less stable than the endogenous S. cerevisiae prion. Heterologous Sup35 proteins coaggregated in the S. cerevisiae cells. However, in vivo cross-species prion conversion was decreased and in vitro polymerization was cross-inhibited in at least some heterologous combinations, thus demonstrating the existence of prion species barrier. Moreover, the barrier between the S. cerevisiae protein and its Saccharomyces paradoxus and Saccharomyces bayanus counterparts was asymmetric both in vivo and in vitro. Our data show that a decreased cross-species prion transmission does not necessarily correlate with a lack of cross-species coaggregation, suggesting that species-specificity of prion transmission is controlled at the level of conformational transition rather than coaggregation.

Triggering receptor expressed on myeloid cell-2 (TREM2) signaling promotes disease-associated microglia (DAM) and phagocytosis in neurodegenerative diseases. Traditional anti-TREM2 agonist antibodies block receptor shedding, lowering soluble TREM2 (sTREM2) and leading to mixed outcomes. We developed 03O05, a ligand-mimetic anti-TREM2 agonist antibody that activates TREM2 while preserving physiological shedding. Binding epitope and cross-reactivity were defined by Bio-Layer Interferometry (BLI) and epitope mapping/mutagenesis. Functional activity was assessed using nuclear factor of activated T cells luciferase reporter (NFAT-luciferase), in vivo DAP12 phosphorylation, and microglial phagocytosis. In vivo effects on sTREM2 levels were evaluated in wild-type (WT), human TREM2 knock-in, and 5xFAD mice by ELISA. Amyloid-beta (Aβ) plaque clearance, microglial state and neuronal health were evaluated in 5xFAD model. Remyelination and microglial status were assessed in the cuprizone model. Anti-TREM2 antibody 03O05 binds a conformational epitope (M41-W44, L89) within the immunoglobulin-like domain, distal from the cleavage site, activates TREM2 signaling in vitro and in vivo, and enhances phagocytosis. A single dose treatment of 03O05 increased sTREM2 in serum and brain of WT and human TREM2 knock-in mice. In 5xFAD mice, chronic 03O05 treatment elevated serum and brain sTREM2, promoted clearance of filamentous Aβ plaques, reduced microgliosis while enhancing microglial phagocytosis, and ameliorated neuronal dystrophy. In the cuprizone model, 03O05 enhanced microglial phagocytosis and promoted remyelination by reducing degraded myelin basic protein (MBP) during recovery. Unlike stalk-binding anti-TREM2 agonist antibodies, 03O05 preserves ectodomain shedding, leading to transient receptor activation and increased sTREM2 levels. This approach promotes a neuroprotective microglial phenotype without inducing neuroinflammation, reduces amyloid pathology and neuronal dystrophy, as well as supports remyelination in multiple sclerosis (MS). These findings suggest the therapeutic potential of shedding-permissive TREM2 agonism in neurodegenerative disease.

The raceway adiabatic flame temperature (RAFT) is the basis for judging the thermal state of the hearth and an important parameter for the blast furnace (BF) operation. However, the traditional model fails to accurately characterize the actual RAFT suitable for H 2 -rich gas injection BF. In this study, a RAFT heat balance model suitable for BF with injection of H 2 -rich gas (shale gas, coke oven gas and H 2 ) was optimized. The influences of the H 2 concentrations in tuyere gases, O 2 enrichment ratio, pulverized coal injection (PCI) quantity and blast humidity on RAFT were calculated and the mathematical formula was set up through multiple linear regression. The results show that with the injection rate of coke oven gas, H 2 and shale gas, the RAFT decreases at a rate of 10.4 ℃ per kg, 14.7 ℃ per kg and 5.92 ℃ per kg, respectively. In addition, RAFT increases with the increase of oxygen enrichment ratio, while decreases with the increase of PCI quantity and blast humidity. Changing the oxygen enrichment ratio, PCI quantity and blast humidity can modulate RAFT when the H 2 -rich gas is injected into BF. This work provides a reference for the H 2 -rich gas injection BF. Graphical Abstract

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via β-arrestin-2 (β-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a β-arr2–mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)–rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates β-arr2–dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal–regulated kinase 1/2 (ERK1/2) activation is also dependent on β-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-β-arr2–mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, β-arr2–driven signaling pathways in caveolae.

BackgroundTwo-dimensional (2D) specimen radiography (SR) and tomosynthesis (DBT) for breast cancer yield data that lack high-depth resolution. A volumetric specimen imager (VSI) was developed to provide full-3D and thin-slice cross-sectional visualization at a 360° view angle. The purpose of this prospective trial was to compare VSI, 2D SR, and DBT interpretation of lumpectomy margin status with the final pathologic margin status of breast lumpectomy specimens.MethodsThe study enrolled 200 cases from two institutions. After standard imaging and interpretation was performed, the main lumpectomy specimen was imaged with the VSI device. Image interpretation was performed by three radiologists after surgery based on VSI, 2D SR, and DBT. A receiver operating characteristic (ROC) curve was created for each method. The area under the curve (AUC) was computed to characterize the performance of the imaging method interpreted by each user.ResultsFrom 200 lesions, 1200 margins were interpreted. The AUC values of VSI for the three radiologists were respectively 0.91, 0.90, and 0.94, showing relative improvement over the AUCs of 2D SR by 54%, 13%, and 40% and DBT by 32% and 11%, respectively. The VSI has sensitivity ranging from 91 to 94%, specificity ranging from 81 to 85%, a positive predictive value ranging from 25 to 30%, and a negative predicative value of 99%.ConclusionsThe ROC curves of the VSI were higher than those of the other specimen imaging methods. Full-3D specimen imaging can improve the correlation between the main lumpectomy specimen margin status and surgical pathology. The findings from this study suggest that using the VSI device for intraoperative margin assessment could further reduce the re-excision rates for women with malignant disease.

Computed tomography (CT) has grown into a major workhorse in radiology since its emergence in the 1970's, for its noninvasiveness, three-dimensional information, and superior contrast resolution. There had been a number of major advances in the CT technology, including optimization-based reconstruction methods, which can be designed to reduce image artifacts and enable flexible scanning configuration design. More recently, there has been a renewed interest in exploring the energy information in CT imaging using multispectral scans. A number of commercial scanners are available to acquire dual-energy scan data for a range of clinical applications. On the other hand, a common limitation shared by almost all commercial dual-energy CT scanners is the significant addition of special hardware to conventional diagnostic CT, adding on to the already-expensive cost of CT systems. Part of the reason for the dependence on the special hardware to acquire dual-energy or multispectral CT data is the need to conform to the data conditions required by the reconstruction methods that include either data-domain or image-domain decomposition and the failure to take advantage of the design flexibility enabled by fully-modeled, optimization-based reconstruction methods, such as the one-step inversion methods for multispectral CT. In this dissertation work, we aim to propose a one-step, optimization-based reconstruction method and enable novel, non-standard scan configurations of potential practical significance for multispectral CT that can be readily implemented on existing conventional CT scanners with no or minimum system modification. We start with the development of the method, including a non-linear data model, a non-convex optimization program, and an algorithm for numerically solving the program, and applied the method to both simulated and real data collected from standard, full-scan and non-standard, partial-scan configurations. The results suggest that fast, low-dose, and low-cost multispectral CT can be enabled by the proposed optimization-based reconstruction and the ASD-NC-POCS algorithm.