Reversible Dual Fluorescence-Lifetime Imaging of Mitochondrial GSH and Microviscosity: Real-Time Evaluation of Ferroptosis Status.

Ferroptosis, as a new form of regulated cell death, is implicated in various physiological and pathological processes. Developing a single probe for an independent analysis of multiple analytes related to ferroptosis can provide more accurate information and simplify the detection procedures, but it faces great challenges. In this work, we develop a fluorescent probe for the simultaneous detection of GSH through ratiometric fluorescence response and microviscosity via a fluorescence lifetime model. Based on the reversible Michael addition reaction between GSH and unsaturated C═C bond, the probe responds reversibly to GSH with a ratiometric fluorescence variation and a fast response time (t1/2 = 4.7 s). At the same time, the probe is sensitive to environmental viscosity by changing its fluorescence lifetimes. The probe was applied to monitor the drug-induced ferroptosis process through both the classical Xc-/GSH/GPX4- and DHODH-mediated defense mechanisms. We hope that the probe will provide a useful molecular tool for the real-time live-cell imaging of GSH dynamics, which is benefit to unveiling related physiological and pathological processes.

Population pharmacokinetic/pharmacodynamic modeling of H018, a selective JAK1 inhibitor, in healthy Chinese volunteers.

H018 is an orally administered, selective, small-molecule inhibitor of Janus kinase 1 (JAK1) made for the treatment of rheumatoid arthritis. A population pharmacokinetic/pharmacodynamic analysis was conducted to characterize the pharmacokinetic and pharmacodynamic profile of H018 and its active metabolite using data from 48 healthy Chinese volunteers who received a single dose of 10-160 mg of H018 in a phase I clinical study. A two-compartment model with delayed absorption and linear elimination adequately described the pharmacokinetic data of H018. The apparent clearance of H018 was estimated to be 39.0 L/h, and triglyceride was identified as a covariate on it. Pharmacokinetic data of the active metabolite could be well described by a two-compartment model with linear elimination. The exposure-effect relationships in terms of pSTAT1 inhibition were well described by a direct response model, with exposure captured by an active moiety that consisted of H018 and its metabolite, weighted by the ratio of in vitro JAK1 inhibitory activity (1.13). The estimated EC50 value for the active moiety is 601 nM. In the simulation using the final model, the inhibitory effect appeared to have reached a plateau in the high-dose groups, with max inhibition rates of 81.42 %, 88.42 %, and 91.89 % for 80, 120, and 160 mg dose groups, respectively. Taken together, this study will provide an instructive reference of dose selection for subsequent clinical trials.

The Efficacy and Safety of Nirmatrelvir/Ritonavir Against COVID-19 in Elderly Patients.

Objective: To assess the key factors influencing the effectiveness of nirmatrelvir/ritonavir in treating elderly patients with COVID-19. Methods: This study was conducted on patients aged ≥60 who were admitted to the Second Affiliated Hospital of Soochow University for COVID-19 infection and were treated with nirmatrelvir/ritonavir. Clinical information was collected from patients and steady-state blood concentrations of nirmatrelvir and ritonavir were measured. Factors associated with treatment effects were searched by univariate and multivariate analysis. Results: A total of 68 (51 males and 17 females) patients had a median age of 80 (73.0-84.8) years were enrolled in this study. The blood concentration measurements (trough concentrations) of nirmatrelvir and ritonavir were 5.1 (2.6-7.1) and 0.4 (0.2-0.9) µg/mL, respectively. Adverse drug reaction was reported in 4 (5.9%) patients. Univariate analysis showed that age, clinical classification, APACHE II score, total bilirubin (TBil), aspartate transaminase (AST), lactate dehydrogenase (LDH), and total cholesterol (TC) were significantly associated with the effectiveness of treatment (P value <0.05). Concentration of nirmatrelvir was also associated with treatment outcome (P value <0.1). Based on the results of univariate analysis, the above factors were introduced into the multiple linear regression equation as independent variables, and the results showed that clinical classification was included in the regression equation model and was the most important factor affecting the treatment outcome. By receiver operating characteristic curve analysis, the area under curve of age + biochemical indicators + APACHE II score + clinical classification was 0.968 (95% CI = 0.919-1.000; P <0.0001). Among the 68 patients included in the study, 4 cases experienced adverse drug reactions. Conclusion: Age, clinical classification, APACHE II score, TBil, AST, LDH, and TC were significantly associated with the effectiveness of treatment in elderly patients with COVID-19.

Population Pharmacokinetic/Pharmacodynamic Models for P2Y12 Inhibitors: A Systematic Review and Clinical Appraisal Using Exposure Simulation.

BACKGROUND AND OBJECTIVE: Recent research indicates a correlation between plasma concentration of P2Y12 inhibitors and clinical events, particularly bleeding, which significantly impeded their clinical therapeutic performance. It is therefore vital to delve into the factors that might affect the plasma concentration. The study aims to summarize population pharmacokinetics/pharmacodynamics (PopPKPD) models for commonly prescribed P2Y12 inhibitors (clopidogrel, prasugrel, and ticagrelor) and assess bleeding risk in specific individual groups. METHODS: The PopPKPD models of P2Y12 inhibitors were collected and summarized based on predetermined inclusion and exclusion criteria. The collected models were replicated in simulations, which were used to assess factors affecting plasma concentrations of P2Y12 inhibitors. Simulation results for special populations were compared to therapeutic window based on reported exposure-effect relationships (PK/PD-related bleeding and thrombotic clinical outcomes) to predict bleeding risk in special populations with different dosing regimens and cumulative covariates. RESULT: Finally, 12 studies were included for PK simulation, 7 of which that also included PD data were subjected to further analysis, with the majority being based on Phase I or II trials. Simulations showed that several covariates such as female gender, weight, elderly can significantly impact on exposure, with special populations reaching up to 179% of the general population. However, after dose adjustment, blood concentrations for special populations can reach approximately ±20% of general population exposure. Therefore, lowering the maintenance dose of ticagrelor from 90 to 60 mg bid was first recommended to reduce bleeding risk without significantly increasing ischemic risk, particularly in elderly, small-weight Asian females. CONCLUSION: Lowering the maintenance dose of ticagrelor from 90 to 60 mg bid effectively reduces bleeding risk without increasing thrombotic infarction risk in elderly, small-weight Asian females.

Role of metabolomic profile as a potential marker to discriminate membranous nephropathy from IgA nephropathy.

BACKGROUND: Membranous nephropathy (MN) and IgA nephropathy (IgAN) are the most common primary glomerulopathies worldwide. The systemic metabolic changes in the progression of MN and IgAN are not fully understood. METHODS: A total of 87 and 70 patients with MN and IgAN, respectively, and 30 healthy controls were enrolled in this study. Untargeted metabolomics was performed to explore the differential metabolites and metabolic pathways in the early stage of MN and IgAN. To judge the diagnostic ability of biomarkers, receiver operating characteristic curve analysis (ROC) were performed. RESULTS: Principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) suggested that patients with MN and IgAN showed an obvious separation trend from the healthy controls. In addition, 155 and 148 metabolites were identified to be significantly altered in the MN and IgAN groups, respectively. Of these, 70 metabolites were markedly altered in both disease groups; six metabolites, including L-tryptophan, L-kynurenine, gamma-aminobutyric acid (GABA), indoleacetaldehyde, 5-hydroxyindoleacetylglycine, and N-alpha-acetyllysine, showed the opposite tendency. The most affected metabolic pathways included the amino acid metabolic pathways, citrate cycle, pantothenate and CoA biosynthesis, and hormone signaling pathways. CONCLUSIONS: Substantial metabolic disorders occurred during the progression of MN and IgAN. L-tryptophan, L-kynurenine, GABA, indoleacetaldehyde, 5-hydroxyindoleacetylglycine, and N-alpha-acetyllysine may show potential as biomarkers for the identification of MN and IgAN.

Systematic review and model-based analysis to identify whether renal safety risks of URAT1 inhibitors are fully determined by uric acid-lowering efficacies.

OBJECTIVE: Renal safety risk is currently an important factor that hinders the development of uric acid transporter 1 (URAT1) inhibitors. This study aimed to compare the renal safety and uric acid-lowering efficacy of different URAT1 inhibitors and clarify the association between them. METHODS: A systematic review of published randomized controlled trials on URAT1 inhibitors was conducted to investigate the incidence of renal safety events. A model-based analysis was performed to predict the uric acid-lowering efficacy of representative URAT1 inhibitors. RESULTS: The overall renal safety event incidences of lesinurad, verinurad, dotinurad, SHR4640, and benzbromarone in patients with hyperuricemia were 11.2 % (142/1264), 12.0 % (34/284), 0.5 % (2/421), 2.3 % (5/213), and 1.3 % (5/393), respectively. A semi-mechanistic pharmacokinetic/pharmacodynamic model was used to establish the dose-exposure-effect relationship of lesinurad, verinurad, dotinurad, and SHR4640 with or without the combination of xanthine oxidase inhibitors (XOIs). The efficacy ranking of the intermediate dose of URAT1 inhibitors with once-daily dosing was 2 mg dotinurad > 10 mg verinurad > 5 mg SHR4640 > 400 mg lesinurad. The combination of 80 mg febuxostat and 600 mg allopurinol reduced the 24-h cumulative renal uric acid excretion by 48.4 % and 48.3 %, respectively. CONCLUSION: Uric acid-lowering efficacy is not an independent factor for the renal safety risk of different URAT1 inhibitors, and structural differences could be responsible for the difference. The adverse renal effects of URAT1 inhibitors are dose-dependent, and the combination with high doses of XOIs can significantly reduce the renal safety risk by reducing uric acid excretion by the kidneys.

Population pharmacokinetic modeling and simulation for nirmatrelvir exposure assessment in Chinese older patients with COVID-19 infection.

Nirmatrelvir is an effective component of Paxlovid, the first oral antiviral drug granted emergency use authorization by the FDA. Nirmatrelvir is prescribed extensively in older adult patients to treat the coronavirus disease 2019 (COVID-19) infection. In this study, population pharmacokinetic modeling with clinical study data was employed to explore the pharmacokinetic profile of nirmatrelvir in older adult Chinese patients with COVID-19 infection. The result suggests that the pharmacokinetic profile of nirmatrelvir can be described by a one-compartment model with first-order absorption and elimination in this study population. The calculated apparent clearance (CL/F), apparent volumes of distribution (V/F), and absorption rate constant (ka) for the typical patient were 4.16 L/h, 39.1 L, and 0.776, respectively. The area under the curve (AUC) of nirmatrelvir in the typical Chinese older adult was approximately three-fold higher than the AUCs in Chinese and Western young adult volunteers. At the same doses, the simulated AUCs were increased by 26%, 43%, 72%, and 135% in virtual populations with creatinine clearances of 60, 45, 30, and 15 mL/min, respectively. Our research provides an instructive reference for nirmatrelvir dose selection in older Chinese adults.

Improving plant heat tolerance through modification of Rubisco activase in C3 plants to secure crop yield and food security in a future warming world.

The world's population may reach 10 billion by 2050, but 10% still suffer from food shortages. At the same time, global warming threatens food security by decreasing crop yields, so it is necessary to develop crops with enhanced resistance to high temperatures in order to secure the food supply. In this review, the role of Rubisco activase as an important factor in plant heat tolerance is summarized, based on the conclusions of recent findings. Rubisco activase is a molecular chaperone determining the activation of Rubisco, whose heat sensitivity causes reductions of photosynthesis at high temperatures. Thus, the thermostability of Rubisco activase is considered to be critical for improving plant heat tolerance. It has been shown that the introduction of thermostable Rubisco activase through gene editing into Arabidopsis thaliana and from heat-adapted wild Oryza species or C4Zea mays into Oryza sativa improves Rubisco activation, photosynthesis, and plant growth at high temperatures. We propose that developing a universal thermostable Rubisco activase could be a promising direction for further studies.

Assessment of Aging-Related Function Variations of P-gp Transporter in Old-Elderly Chinese CHF Patients Based on Modeling and Simulation.

BACKGROUND AND OBJECTIVES: P-glycoprotein (P-gp) is one of the most intensely studied transporters owing to its broad tissue distribution and substrate specificity. Existing research suggests that the risk of systemic exposure to dabigatran etexilate (DABE) and digoxin, two P-gp probe substrates in vivo, has significantly increased in elderly patients. We applied a model-based quantitative pharmacological approach to assess aging-related P-gp changes in the Chinese old-elderly population. METHODS: Population pharmacokinetic (PopPK) modeling was first performed using clinical pharmacokinetic data to explore the effect of age on the pharmacokinetic characteristics of dabigatran (DAB, the active principle of DABE) and digoxin in elderly Chinese patients. Corresponding physiologically based pharmacokinetic (PBPK) models were established to further explain the elevated systemic exposure to these two drugs. Eventually, standard dosing regimens of DABE and digoxin were assessed in Chinese old-elderly patients with chronic heart failure (CHF) with different stages of renal impairment. RESULTS: PopPK analysis suggested that age as a covariate had an additional effect on the apparent clearance of these two drugs after correcting for creatinine clearance. PBPK simulation results suggested that disease-specific pathophysiological changes could explain DAB exposure in the young elderly. In the elderly population, 17.1% of elevated DAB exposure remained unexplained, and 25.5% of the reduced P-gp function associated with aging was ultimately obtained using sensitivity analysis. This value was further validated using digoxin data obtained by PBPK modeling. The simulation results suggest that CHF patients with advanced age and moderate-to-severe renal impairment require heightened vigilance for elevated exposure risk during the use of DABE and digoxin. CONCLUSIONS: Aging might be a significant risk factor for elevated systemic exposure to DAB and digoxin by reducing P-gp-mediated efflux in the Chinese old elderly population.

Anti-proliferation and apoptosis-inducing effects of dihydroartemisinin on SH-SY5Y cells and metabolomic analysis.

Background: In childhood, metastatic neuroblastoma (NB) is the most common extracranial solid tumor, but there are no appropriate drugs for its treatment. Dihydroartemisinin (DHA), a drug for malaria treatment, has therapeutic potential in several cancers; however, its mechanisms remain unclear. This study aimed to investigate the anti-proliferation effect of DHA on SH-SY5Y cells and to explore its mechanism in vitro. Methods: We used 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to measure the half-maximal inhibitory concentration (IC50) of DHA; western blot was used to determine protein levels; propidium iodide (PI) staining was used to determine apoptotic cells; JC-1 staining to measure mitochondrial membrane potential; and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining was used to determine reactive oxygen species (ROS). Metabonomic analysis was performed by using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based untargeted metabolomics. Multivariate statistical analysis was performed to screen potential metabolites associated with DHA treatment in SH-SY5Y cells. Results: It was shown that DHA inhibited SH-SY5Y cell proliferation and increased poly (ADP-ribose) polymerase (PARP-1) and caspase 3 in a dose-dependent manner. In Further, DHA promoted ROS generation and γH2AX expression. In addition, a total of 125 proposed metabolites in SH-SY5Y cells and 45 vital metabolic pathways were identified through UHPLC-MS/MS-based untargeted metabolomic analysis. Conclusions: These data suggest that DHA could regulate taurine, linoleic acid, phenylalanine metabolism, and tryptophan metabolism, which are involved in the anti-proliferation effect of DHA in SH-SY5Y cells.

Establishment and validation of analytical methods for 15 hazardous drugs by UPLC-Q/Orbitrap-HRMS.

Background: Cytotoxic drug residues in pharmacy intravenous admixture services (PIVAS) have always been a major problem for pharmaceutical workers and the PIVAS environment,which is not only pollutes the PIVAS environment, but also causes serious harm to the life and health of the staff. This study aimed to establish an ultra-high performance liquid chromatography quadrupole orbitrap high resolution mass spectrometry (UPLC-Q/Orbitrap-HRMS) method for the rapid detection and monitor of 15 cytotoxic drugs. Methods: UPLC-Q/Orbitrap-HRMS method was used to establish a rapid detection method for 15 cytotoxic drugs such as cytarabine, gemcitabine and so on. The daily precision and accuracy of this method were verified by injecting four concentrations of standard solution on the same day, and the same four concentrations of standard solution were injected within three days respectively to verify the daily precision of this method. The signal-to-noise ratio (SNR) of 10:1 was calculated as the limit of quantity. The mixed standard solution of 15 cytotoxic drugs with concentrations of 0.5, 1, 3, 10, 30, 100, 300, and 1,000 ng/mL was configured and detected by this method for linearity and range.The stability of this method was investigated using a mixture of 15 drugs (15MIX) standard solutions at high concentration (300 ng/mL) and low concentration (10 ng/mL) at room temperature for 12 and 24 hours, respectively. A standard solution of each drug, 15MIX and blank solution were taken to verify the exclusivity of the method. Results: The results showed that the method had good specificity, and the intraday precision of all drugs was less than 10% and the intraday precision was less than 15%. At the same time, the standard curve had good linearity, R2 was greater than 0.99, and the limit of quantification of most drugs was about 1 ng/mL. Conclusions: In this study, an UPLC-Q/Orbitrap-HRMS method was established for the rapid detection of 15 cytotoxic drugs, providing technical support for the monitoring of cytotoxic drug residues in PIVAS, which is of great significance for environmental contamination mornitoring as well as occupational exposure alert.

Gut Microbiota-Mediated Elevated Production of Secondary Bile Acids in Chronic Unpredictable Mild Stress.

A growing body of evidence suggests that gut microbiota could participate in the progression of depression via the microbiota-gut-brain axis. However, the detailed microbial metabolic profile changes in the progression of depression is still not fully elucidated. In this study, a liquid chromatography coupled to mass spectrometry-based untargeted serum high-throughput metabolomics method was first performed to screen for potential biomarkers in a depressive-like state in a chronic unpredictable mild stress (CUMS)-induced mouse model. Our results identified that the bile acid and energy metabolism pathways were significantly affected in CUMS progression. The detailed bile acid profiles were subsequently quantified in the serum, liver, and feces. The results showed that CUMS significantly promoted the deconjugation of conjugated bile acid and secondary bile acid biosynthesis. Furthermore, 16S rRNA gene sequencing revealed that the increased secondary bile acid levels in the feces positively correlated with Ruminococcaceae_UCG-010, Ruminococcus, and Clostridia_UCG-014 abundance. Taken together, our study suggested that changes in family Ruminococcaceae abundance following chronic stress increased biosynthesis of deoxycholic acid (DCA), a unconjugated secondary bile acid in the intestine. Aberrant activation of secondary bile acid biosynthesis pathway thereby increased the hydrophobicity of the bile acid pool, which might, in turn, promoted metabolic disturbances and disease progression in CUMS mice.

Comparative effect of ciprofloxacin and moxifloxacin on the modulation of bile acid profiles and gut microbiota in rats

Abstract Fluoroquinolones are an important class of antimicrobial agents to manage infectious diseases. However, knowledge about how host bile acids are modified by fluoroquinolones is limited. We investigated and compared the impact of fluoroquinolones on circulating bile acid profiles and gut microbiota from in vivo studies. We administered ciprofloxacin (100 mg/kg/day) or moxifloxacin (40 mg/kg/day) orally to male Wistar rats for seven days. Fifteen bile acids (BAs) from the serum and large intestine were quantified by HPLC-MS/MS. The diversity of gut microbiota after ciprofloxacin and moxifloxacin treatment was analyzed using high-throughput, next-generation sequencing technology. The two fluoroquinolone-treated groups had different BA profiles. Ciprofloxacin significantly reduced the hydrophobicity index of the BA pool, reduced secondary BAs, and increased taurine-conjugated primary BAs in both the serum and large intestine as compared with moxifloxacin. Besides, ciprofloxacin treatment altered intestinal microbiota with a remarkable increase in Firmicutes to Bacteroidetes ratio, while moxifloxacin exerted no effect. What we found suggests that different fluoroquinolones have a distinct effect on the host BAs metabolism and intestinal bacteria, and therefore provide guidance on the selection of fluoroquinolones to treat infectious diseases.

Overexpression of both Rubisco and Rubisco activase rescues rice photosynthesis and biomass under heat stress.

Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially Rubisco activation. We examined whether co-overexpression of Rubisco and Rubisco activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines-the wild-type (WT), a Rubisco activase-overexpressing line (oxRCA) and a Rubisco- and Rubisco activase-co-overexpressing line (oxRCA-RBCS)-and analysed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the Rubisco activase content was 153% higher in oxRCA and 138% higher in oxRCA-RBCS, and the Rubisco content was 27% lower in oxRCA and similar in oxRCA-RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to Rubisco deactivation by heat. On the other hand, that of oxRCA and oxRCA-RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA-RBCS was 26% higher than that of WT at 40°C. These results show that increasing the Rubisco activase content without the reduction of Rubisco content could improve yield and sustainability in rice at high temperature.