Enzyme-Inspired Ligand Engineering of Gold Nanoclusters for Electrocatalytic Microenvironment Manipulation.

Natural enzymes intricately regulate substrate accessibility through specific amino acid sequences and folded structures at their active sites. Achieving such precise control over the microenvironment has proven to be challenging in nanocatalysis, especially in the realm of ligand-stabilized metal nanoparticles. Here, we use atomically precise metal nanoclusters (NCs) as model catalysts to demonstrate an effective ligand engineering strategy to control the local concentration of CO2 on the surface of gold (Au) NCs during electrocatalytic CO2 reduction reactions (CO2RR). The precise incorporation of two 2-thiouracil-5-carboxylic acid (TCA) ligands within the pocket-like cavity of [Au25(pMBA)18]- NCs (pMBA = para-mercaptobenzoic acid) leads to a substantial acceleration in the reaction kinetics of CO2RR. This enhancement is attributed to a more favorable microenvironment in proximity to the active site for CO2, facilitated by supramolecular interactions between the nucleophilic Nδ- of the pyrimidine ring of the TCA ligand and the electrophilic Cδ+ of CO2. A comprehensive investigation employing absorption spectroscopy, mass spectrometry, isotopic labeling measurements, electrochemical analyses, and quantum chemical computation highlights the pivotal role of local CO2 enrichment in enhancing the activity and selectivity of TCA-modified Au25 NCs for CO2RR. Notably, a high Faradaic efficiency of 98.6% toward CO has been achieved. The surface engineering approach and catalytic fundamentals elucidated in this study provide a systematic foundation for the molecular-level design of metal-based electrocatalysts.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

The detection of temperature by the human sensory system is life-preserving and highly evolutionarily conserved. Platelets are sensitive to temperature changes and are activated by a decrease in temperature, akin to sensory neurons. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this multidisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

When polyethylene terephthalate microplastics meet Perfluorooctane sulfonate in thermophilic biogas upgrading system: Their effect on methanogenesis.

Microplastics (MPs) and Perfluorooctane sulfonate (PFOS) are two hard-biodegradable pollutants widely existing in the waste streams treated by anaerobic digestion. However, their synergistic effect on methanogenic metabolism is still unknown. This study investigated the impact of polyethylene terephthalate (PET) MPs alone and co-existing with PFOS on CO2 conversion to CH4 in a thermophilic biogas upgrading system. The results showed that either PET MPs addition alone or coexisting with PFOS improved the ultimate CH4 percentage and increased CO2 utilization rate. When Fe0 was added into the reactors with PET to enhance the interspecies electron transfer, a potential defluorination was observed with a defluorination rate of 15.88 ± 1.53%. Exposure of the reactor to PFOS of 300 µg/L could change the methanogenic pathway, resulting in a newly emerged Methanomassiliicoccus with dominance of 16%. Furthermore, under the exposure of PFOS, the number of predicted genes regulating enzymes in methanogenic steps from CO2 increased. These results suggest that the co-existence of PET MPs and PFOS will not inhibit the activity of hydrotrophic methanogenes, and a portion of PFOS may be biodegraded during the methanogenesis under Fe0 regulation.

Distinct platelet F-actin patterns and traction forces on von Willebrand factor versus fibrinogen.

Upon vascular injury, platelets form a hemostatic plug by binding to the subendothelium and to each other. Platelet-to-matrix binding is initially mediated by von Willebrand factor (VWF) and platelet-to-platelet binding is mediated mainly by fibrinogen and VWF. After binding, the actin cytoskeleton of a platelet drives its contraction, generating traction forces that are important to the cessation of bleeding. Our understanding of the relationship between adhesive environment, F-actin morphology, and traction forces is limited. Here, we examined F-actin morphology of platelets attached to surfaces coated with fibrinogen and VWF. We identified distinct F-actin patterns induced by these protein coatings and found that these patterns were identifiable into three classifications via machine learning: solid, nodular, and hollow. We observed that traction forces for platelets were significantly higher on VWF than on fibrinogen coatings and these forces varied by F-actin pattern. In addition, we analyzed the F-actin orientation in platelets and noted that their filaments were more circumferential when on fibrinogen coatings and having a hollow F-actin pattern, while they were more radial on VWF and having a solid F-actin pattern. Finally, we noted that subcellular localization of traction forces corresponded to protein coating and F-actin pattern: VWF-bound, solid platelets had higher forces at their central region while fibrinogen-bound, hollow platelets had higher forces at their periphery. These distinct F-actin patterns on fibrinogen and VWF and their differences in F-actin orientation, force magnitude, and force localization could have implications in hemostasis, thrombus architecture, and venous versus arterial thrombosis.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

Platelets are sensitive to temperature changes and akin to sensory neurons, are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this interdisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Low-density lipoprotein promotes microvascular thrombosis by enhancing von Willebrand factor self-association.

von Willebrand factor (VWF) mediates primary hemostasis and thrombosis in response to hydrodynamic forces. We previously showed that high shear promoted self-association of VWF into hyperadhesive strands, which can be attenuated by high-density lipoprotein (HDL) and apolipoprotein A-I. In this study, we show that low-density lipoprotein (LDL) binds VWF under shear and enhances self-association. Vortexing VWF in tubes resulted in its loss from the solution and deposition onto tube surfaces, which was prevented by HDL. At a stabilizing HDL concentration of 1.2 mg/mL, increasing concentrations of LDL progressively increased VWF loss, the effect correlating with the LDL-to-HDL ratio and not the absolute concentration of the lipoproteins. Similarly, HDL diminished deposition of VWF in a post-in-channel microfluidic device, whereas LDL increased both the rate and extent of strand deposition, with both purified VWF and plasma. Hypercholesterolemic human plasma also displayed accelerated VWF accumulation in the microfluidic device. The initial rate of accumulation correlated linearly with the LDL-to-HDL ratio. In Adamts13-/- and Adamts13-/-LDLR-/- mice, high LDL levels enhanced VWF and platelet adhesion to the myocardial microvasculature, reducing cardiac perfusion, impairing systolic function, and producing early signs of cardiomyopathy. In wild-type mice, high plasma LDL concentrations also increased the size and persistence of VWF-platelet thrombi in ionophore-treated mesenteric microvessels, exceeding the accumulation seen in similarly treated ADAMTS13-deficient mice that did not receive LDL infusion. We propose that targeting the interaction of VWF with itself and with LDL may improve the course of thrombotic microangiopathies, atherosclerosis, and other disorders with defective microvascular circulation.

Promote electroreduction of CO2 via catalyst valence state manipulation by surface-capping ligand.

Electrochemical CO2 reduction provides a potential means for synthesizing value-added chemicals over the near equilibrium potential regime, i.e., formate production on Pd-based catalysts. However, the activity of Pd catalysts has been largely plagued by the potential-depended deactivation pathways (e.g., [Formula: see text]-PdH to [Formula: see text]-PdH phase transition, CO poisoning), limiting the formate production to a narrow potential window of 0 V to -0.25 V vs. reversible hydrogen electrode (RHE). Herein, we discovered that the Pd surface capped with polyvinylpyrrolidone (PVP) ligand exhibits effective resistance to the potential-depended deactivations and can catalyze formate production at a much extended potential window (beyond -0.7 V vs. RHE) with significantly improved activity (~14-times enhancement at -0.4 V vs. RHE) compared to that of the pristine Pd surface. Combined results from physical and electrochemical characterizations, kinetic analysis, and first-principle simulations suggest that the PVP capping ligand can effectively stabilize the high-valence-state Pd species (Pdδ+) resulted from the catalyst synthesis and pretreatments, and these Pdδ+ species are responsible for the inhibited phase transition from [Formula: see text]-PdH to [Formula: see text]-PdH, and the suppression of CO and H2 formation. The present study confers a desired catalyst design principle, introducing positive charges into Pd-based electrocatalyst to enable efficient and stable CO2 to formate conversion.

Plasma Interleukin-6 (IL-6), Angiopoietin-2, and C-Reactive Protein Levels Predict Subsequent Type 1 Myocardial Infarction in Persons With Treated HIV Infection.

BACKGROUND: HIV infection leads to endothelial activation, promoting platelet adhesion, and accelerating atherosclerosis. Our goal was to determine whether biomarkers of endothelial activation and hemostasis/thrombosis were elevated in people with treated HIV (PWH) before myocardial infarction (MI). METHODS: In a case-control study nested within the CFAR Network of Integrated Clinical Systems (CNICS) cohort, we compared 69 adjudicated cases with type 1 MI with 138 controls matched for antiretroviral therapy regimen. We measured angiopoietin-1, angiopoietin-2 (ANG-2), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), von Willebrand factor, C-reactive protein (CRP), interleukin-6 (IL-6), plasminogen activation inhibitor-1, P-selectin, serum amyloid-A, soluble CD14, and apolipoprotein A1 in stored plasma. Conditional logistic regression identified associations with subsequent MI, with and without adjustment for Atherosclerotic Cardiovascular Disease (ASCVD) and Veterans Aging Cohort Study (VACS) scores. RESULTS: Higher IL-6 was associated with MI after adjustment for ASCVD score (adjusted odds ratio [AOR] 1.51, 95% confidence interval [95% CI]: 1.05 to 2.17 per standard-deviation-scaled log 2 increment). In a separate model adjusting for VACS score, higher ANG-2 (AOR 1.49, 95% CI: 1.04 to 2.14), higher CRP (AOR 1.45, 95% CI: 1.06 to 2.00), and higher IL-6 (AOR 1.68, 95% CI: 1.17 to 2.41) were associated with MI. In a sensitivity analysis excluding PWH with viral load ≥400 copies/mL, higher IL-6 remained associated with MI after adjustment for ASCVD score and after adjustment for VACS score. CONCLUSIONS: Among PWH, higher levels of plasma IL-6, CRP, and ANG-2 predict subsequent type 1 MI, independent of conventional risk scores. IL-6 had the most consistent associations with type 1 MI, regardless of viral load suppression.

Increased Susceptibility for Adverse Reactions to Ultrasound Enhancing Agents in Sickle Cell Disease.

BACKGROUND: Pain-related adverse events (AEs) to ultrasound enhancing agents (UEAs) have been reported in patients with sickle cell disease (SCD). The aims of this study were to characterize the scope of these AEs in the SCD population and to investigate potential mechanisms on the basis of pathways involved in SCD vaso-occlusive crisis (VOC) and pain. METHODS: The prevalence and classification of AEs were analyzed from two clinical trials in which high-dose Definity infusions were used in patients with SCD (n = 55) or matched control subjects (n = 43) to study muscle or myocardial microvascular perfusion. Because complement (C') activation can trigger VOC in SCD, C' activation and surface adhesion of C' proteins on lipid UEAs were studied in vitro. C'-mediated UEA attachment to bone marrow immune cells was assessed using flow cytometry in a murine SCD model (Townes mice). Blood from patients receiving Definity was obtained to measure specific lysophospholipid metabolites of lipids in Definity thought to mediate SCD pain. RESULTS: Moderate or greater AEs, all of which were nociceptive (back or bone pain), occurred in one control subject and nine SCD subjects (2% vs 16%, P = .02). Patients with SCD who had AEs tended to have more severe manifestations of SCD. Three of the subjects with SCD had previously received Definity without complications. In patients with SCD, four AEs were classified as severe in intensity and as serious AEs on the basis of need for medical intervention. AEs were described to be similar to SCD-related pain, but there was no evidence for VOC, hemolysis, hypotension, or hypoxemia. At baseline, markers of C' activation were greater in patients with SCD than control subjects. However, after administration of lipid UEAs, SCD and control subjects were similar with regard to C' activation response, anaphylatoxin production, bone marrow microbubble retention, and production of lysophospholipids. There was a trend toward increased deposition of C3b and C3bi on lipid UEAs exposed to serum from patients with SCD. CONCLUSIONS: Patients with SCD are particularly susceptible to nociceptive AEs when given Definity at high doses. The mechanism for these AEs remains unclear but most are not related to the triggering of classic VOC.

The Interplay between Meningeal Lymphatic Vessels and Neuroinflammation in Neurodegenerative Diseases.

Meningeal lymphatic vessels (MLVs) are essential for the drainage of cerebrospinal fluid, macromolecules, and immune cells in the central nervous system. They play critical roles in modulating neuroinflammation in neurodegenerative diseases. Dysfunctional MLVs have been demonstrated to increase neuroinflammation by horizontally blocking the drainage of neurotoxic proteins to the peripheral lymph nodes. Conversely, MLVs protect against neuroinflammation by preventing immune cells from becoming fully encephalitogenic. Furthermore, evidence suggests that neuroinflammation affects the structure and function of MLVs, causing vascular anomalies and angiogenesis. Although this field is still in its infancy, the strong link between MLVs and neuroinflammation has emerged as a potential target for slowing the progression of neurodegenerative diseases. This review provides a brief history of the discovery of MLVs, introduces in vivo and in vitro MLV models, highlights the molecular mechanisms through which MLVs contribute to and protect against neuroinflammation, and discusses the potential impact of neuroinflammation on MLVs, focusing on recent progress in neurodegenerative diseases.

Pinpointing the axial ligand effect on platinum single-atom-catalyst towards efficient alkaline hydrogen evolution reaction.

Developing active single-atom-catalyst (SAC) for alkaline hydrogen evolution reaction (HER) is a promising solution to lower the green hydrogen cost. However, the correlations are not clear between the chemical environments around the active-sites and their desired catalytic activity. Here we study a group of SACs prepared by anchoring platinum atoms on NiFe-layered-double-hydroxide. While maintaining the homogeneity of the Pt-SACs, various axial ligands (-F, -Cl, -Br, -I, -OH) are employed via a facile irradiation-impregnation procedure, enabling us to discover definite chemical-environments/performance correlations. Owing to its high first-electron-affinity, chloride chelated Pt-SAC exhibits optimized bindings with hydrogen and hydroxide, which favor the sluggish water dissociation and further promote the alkaline HER. Specifically, it shows high mass-activity of 30.6 A mgPt-1 and turnover frequency of 30.3 H2 s-1 at 100 mV overpotential, which are significantly higher than those of the state-of-the-art Pt-SACs and commercial Pt/C catalyst. Moreover, high energy efficiency of 80% is obtained for the alkaline water electrolyser assembled using the above catalyst under practical-relevant conditions.

The impact of self-compassionate mindfulness on online learning behavioral engagement of international students during COVID-19: Positive emotion and self-improvement motivation as mediators.

Focusing on the domain of self-compassion, this study explored the promotion mechanism of online learning behavioral engagement (OLBE) of international students in China under COVID-19. Positive emotion and self-improvement motivation were selected as mediators. Participants were 606 international students from 8 countries who were studying online in their own countries due to the international travel restriction of COVID-19. Results showed positive emotion and self-improvement motivation completely mediated self-compassionate mindfulness (SCM) and OLBE of international students. Positive emotion and self-improvement partially mediated SCM and OLBE of international students respectively. Students with higher SCM engage with online learning more in that they possess more positive emotion and self-improvement motivation. This study suggested that SCM may facilitate OLBE via positive emotion and effective self-improvement motivation.

Functionalized Ag with Thiol Ligand to Promote Effective CO2 Electroreduction.

It is challenging while critical to develop efficient catalysts that can achieve both high current density and high energy efficiency for electrocatalytic CO2 reduction (CO2R). Herein, we report a strategy of tailoring the surface electronic structure of an Ag catalyst via thiol ligand modification to improve its intrinsic activity, selectivity, and further energy efficiency toward CO2R. Specifically, interconnected Ag nanoparticles with residual thiol ligands on the surface were prepared through electrochemical activation of a thiol-ligand-based Ag complex. When it was used as a catalyst for CO2R, the thiol-ligand modified Ag exhibited high CO selectivity (>90%) throughout a wide electrode-potential range; furthermore, high cathodic energy efficiencies of >90% and >70% were obtained for CO formation at high current densities of 150 and 750 mA cm-2, respectively, outperforming the state-of-the-art Ag-based electrocatalysts for CO2 to CO conversion. The first-principle calculations on the reaction energetics suggest that the binding energies of the key intermediate -*COOH on Ag are optimized by the adsorbed thiol ligand, thus favoring CO formation while suppressing the competing H2 evolution. Our findings provide a rational design strategy for CO2 reduction electrocatalyst by electronic modulation through surface-adsorbed ligands.

Reduced Proteolytic Cleavage of von Willebrand Factor Leads to Aortic Valve Stenosis and Load-Dependent Ventricular Remodeling.

We hypothesized that excess endothelial-associated von Willebrand factor (vWF) and secondary platelet adhesion contribute to aortic valve stenosis (AS). We studied hyperlipidemic mice lacking ADAMTS13 (LDLR -/- AD13 -/- ), which cleaves endothelial-associated vWF multimers. On echocardiography and molecular imaging, LDLR -/- AD13 -/- compared with control strains had increased aortic endothelial vWF and platelet adhesion and developed hemodynamically significant AS, arterial stiffening, high valvulo-aortic impedance, and secondary load-dependent reduction in LV systolic function. Histology revealed leaflet thickening and calcification with valve interstitial cell myofibroblastic and osteogenic transformation, and evidence for TGFß1 pathway activation. We conclude that valve leaflet endothelial vWF-platelet interactions promote AS through juxtacrine platelet signaling.

Endothelial-derived von Willebrand factor accelerates fibrin clotting within engineered microvessels.

BACKGROUND: Von Willebrand factor (VWF) is classically associated with primary hemostasis and platelet-rich arterial thromboses, but recently has also been implicated in fibrin clotting and venous thrombosis. Direct interaction between fibrin and VWF may mediate these processes, although prior reports are conflicting. OBJECTIVES: We combined two complementary platforms to characterize VWF-fibrin(ogen) interactions and identify their potential physiologic significance. METHODS: Engineered microvessels were lined with human endothelial cells, cultured under flow, and activated to release VWF and form transluminal VWF fibers. Fibrinogen, fibrin monomers, or polymerizing fibrin were then perfused, and interactions with VWF evaluated. Thrombin and fibrinogen were perfused into living versus paraformeldahyde-fixed microvessels and the pressure drop across microvessels monitored. Separately, protein binding to tethered VWF was assessed on a single-molecule level using total internal reflection fluorescence (TIRF) microscopy. RESULTS: Within microvessels, VWF fibers colocalized with polymerizing fibrin, but not fibrinogen. TIRF microscopy showed no colocalization between VWF and fibrinogen or fibrin monomers in a microfluidic flow chamber across a range of shear rates and protein concentrations. Thrombin-mediated fibrin polymerization within living microvessels triggered endothelial VWF release, increasing the rate and amount of microvessel obstruction compared to fixed vessels with an inert endothelium. CONCLUSIONS: We did not identify specific binding between fibrin(ogen) and VWF at a single-molecule level. Despite this, our results suggest that rapid release of endothelial VWF during clotting may provide a physical support for fibrin polymerization and accelerate thrombosis. This interaction may be of fundamental importance for the understanding and treatment of human thrombotic disease.

Deletion of platelet CLEC-2 decreases GPIbα-mediated integrin αIIbß3 activation and decreases thrombosis in TTP.

Microvascular thrombosis in patients with thrombotic thrombocytopenic purpura (TTP) is initiated by GPIbα-mediated platelet binding to von Willebrand factor (VWF). Binding of VWF to GPIbα causes activation of the platelet surface integrin αIIbß3. However, the mechanism of GPIbα-initiated activation of αIIbß3 and its clinical importance for microvascular thrombosis remain elusive. Deletion of platelet C-type lectin-like receptor 2 (CLEC-2) did not prevent VWF binding to platelets but specifically inhibited platelet aggregation induced by VWF binding in mice. Deletion of platelet CLEC-2 also inhibited αIIbß3 activation induced by the binding of VWF to GPIbα. Using a mouse model of TTP, which was created by infusion of anti-mouse ADAMTS13 monoclonal antibodies followed by infusion of VWF, we found that deletion of platelet CLEC-2 decreased pulmonary arterial thrombosis and the severity of thrombocytopenia. Importantly, prophylactic oral administration of aspirin, an inhibitor of platelet activation, and therapeutic treatment of the TTP mice with eptifibatide, an integrin αIIbß3 antagonist, reduced pulmonary arterial thrombosis in the TTP mouse model. Our observations demonstrate that GPIbα-mediated activation of integrin αIIbß3 plays an important role in the formation of thrombosis in TTP. These observations suggest that prevention of platelet activation with aspirin may reduce the risk for thrombosis in patients with TTP.

Phenotypic analysis of erythrocytes in sickle cell disease using imaging flow cytometry.

The morphology and other phenotypic characteristics of erythrocytes in sickle cell disease (SCD) have been analyzed for decades in patient evaluation. This involves a variety of techniques, including microscopic analysis of stained blood films, flow cytometry, and cell counting. Here, we analyzed SCD blood using imaging flow cytometry (IFC), a technology that combines flow cytometry and microscopy to enable simultaneous rapid-throughput analysis of cellular morphology and cell-surface markers. With IFC, we were able to automate quantification of poikilocytes from SCD blood. An important subpopulation of poikilocytes represented dense cells, although these could not be distinguished from other poikilocytes without first centrifuging the blood through density gradients. In addition, CD71-positive RBCs from SCD patients had two subpopulations: one with high CD71 expression and a puckered morphology and another with lower CD71 expression and biconcave morphology and presumably representing a later stage of differentiation. Some RBCs with puckered morphologies that were strongly positive for DAPI and CD49d were in fact nucleated RBCs. IFC identified more phosphatidylserine-expressing red cells in SCD than did conventional flow cytometry and these could also be divided into two subpopulations. One population had diffuse PS expression and appeared to be composed primarily of RBC ghosts; the other had lower overall PS expression present in intense, punctate dots overlying Howell-Jolly bodies. This study demonstrates that IFC can rapidly reveal and quantify RBC features in SCD that require numerous tedious methods to identify conventionally. Thus, IFC is likely to be a useful technique for evaluating and monitoring SCD.

Screening and verification of CYP3A4 inhibitors from Bushen-Yizhi formula to enhance the bioavailability of osthole in rat plasma.

ETHNOPHARMACOLOGICAL RELEVANCE: With the features of multiple-components and targets as well as multifunction, traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of various diseases for a long time. During the application of TCM, the researches about bioavailability enhancement of the bioactive constituents in formula are flourishing. Bushen-Yizhi formula (BSYZ), a TCM prescription with osthole (OST) as one of the main bioactive ingredients, have been widely used to treat kidney deficiency, mental retardation and Alzheimer's disease. However, the underlying biological mechanism and compound-enzyme interaction mediated bioavailability enhancement of OST are still not clearly illuminated. AIM OF THE STUDY: The aim of this study is to explore the material basis and molecular mechanism from BSYZ in the bioavailability enhancement of OST. Screening the potential CYP3A4 inhibitors using theoretical prediction and then verifying them in vitro, and pharmacokinetics study of OST in rat plasma under co-administrated of screened CYP3A4 inhibitors and BSYZ were also scarcely reported. MATERIALS AND METHODS: Screening of CYP3A4 inhibitors from BSYZ was performed with molecular docking simulation from systems pharmacology database. The screened compounds were verified by using P450-Glo Screening Systems. A multiple reaction monitoring (MRM) mass spectrometry method was established for OST quantification. Male Sprague-Dawley rats divided into four groups and six rats in each group were employed in the pharmacokinetics study of OST. The administrated conditions were group I, OST (20 mg/kg); group II, BSYZ (containing OST 1 mg/mL, at the dose of 20 mg/kg OST in BSYZ); group III, co-administration of ketoconazole (Ket, 75 mg/kg) and OST (20 mg/kg); group IV, co-administration of CYP3A4 inhibitor (10 mg/kg) and OST (20 mg/kg). They were determined by using HPLC-MS/MS (MRM) and statistical analysis was performed using student's t-test with p < 0.05 as the level of significance. RESULTS: 21 potential CYP3A4 inhibitors were screened from BSYZ compounds library. From the results of verification in vitro, we found 4 compounds with better CYP3A4 inhibition efficiency including Oleic acid, 1,2,3,4,6-O-Pentagalloylglucose, Rutin, and Schisantherin B. Under further verification, Schisantherin B exhibited the best inhibitory effect on CYP3A4 (IC50 = 0.339 µM), and even better than the clinically used drug (Ket) at the concentration of 5 µM. In the study of pharmacokinetics, the area under the curve (AUC, ng/L*h) of OST after oral administration of BSYZ, Ket and Schisantherin B (2196.23 ± 581.33, 462.90 ± 92.30 and 1053.03 ± 263.62, respectively) were significantly higher than that of pure OST treatment (227.89 ± 107.90, p < 0.01). CONCLUSIONS: Schisantherin B, a profoundly effective CYP3A4 inhibitor screened from BSYZ antagonized the metabolism of CYP3A4 on OST via activity inhibition, therefore significantly enhanced the bioavailability of OST in rat plasma. The results of this study will be helpful to explain the rationality of the compatibility in TCM formula, and also to develop new TCM formula with more reasonable drug compatibility.

Insights into oral bioavailability enhancement of therapeutic herbal constituents by cytochrome P450 3A inhibition.

Herbal plants typically have complex compositions and diverse mechanisms. Among them, bioactive constituents with relatively high exposure in vivo are likely to exhibit therapeutic efficacy. On the other hand, their bioavailability may be influenced by the synergistic effects of different bioactive components. Cytochrome P450 3A (CYP3A) is one of the most abundant CYP enzymes, responsible for the metabolism of 50% of approved drugs. In recent years, many therapeutic herbal constituents have been identified as CYP3A substrates. It is more evident that CYP3A inhibition derived from the herbal formula plays a critical role in improving the oral bioavailability of therapeutic constituents. CYP3A inhibition may be the mechanism of the synergism of herbal formula. In this review, we explored the multiplicity of CYP3A, summarized herbal monomers with CYP3A inhibitory effects, and evaluated herb-mediated CYP3A inhibition, thereby providing new insights into the mechanisms of CYP3A inhibition-mediated oral herb bioavailability.

Prognostic Biomarkers for Thrombotic Microangiopathy after Acute Graft-versus-Host Disease: A Nested Case-Control Study.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a complication of allogeneic hematopoietic cell transplantation (HCT) that often occurs following the development of acute graft-versus-host disease (aGVHD). In this study, we aimed to identify early TMA biomarkers among patients with aGVHD. We performed a nested-case-control study from a prospective cohort of allogeneic HCT recipients, matching on the timing and severity of antecedent aGVHD. We identified 13 TMA cases and 25 non-TMA controls from 208 patients in the cohort. Using multivariable conditional logistic regression, the odds ratio for TMA compared with non-TMA was 2.65 (95% confidence interval [CI], 1.00 to 7.04) for every 100 ng/mL increase in terminal complement complex sC5b9 and 2.62 (95% CI, 1.56 to 4.38) for every 1000 pg/mL increase in angiopoietin-2 (ANG2) at the onset of aGVHD. ADAMTS13 and von Willebrand factor (VWF) antigens were not appreciably associated with TMA. Using a Cox regression model incorporating sC5b9 >300 ng/mL and ANG2 >3000 pg/mL at the onset of aGVHD, the adjusted hazard ratio for mortality was 5.33 (95% CI, 1.57 to 18.03) for the high-risk group (both elevated) and 4.40 (95% CI, 1.60 to 12.07) for the intermediate-risk group (one elevated) compared with the low-risk group (neither elevated). In conclusion, we found that elevated sC5b9 and ANG2 levels at the onset of aGVHD were associated with the development of TMA and possibly mortality after accounting for the timing and severity of aGVHD. The results suggest important roles of complement activation and endothelial dysfunction in the pathogenesis of TMA. Measurement of these biomarkers at the onset of aGVHD may inform prognostic enrichment for preventive trials and improve clinical care.