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Background: Glycoprotein non-metastatic melanoma B (GPNMB)/osteoactivin was first identified in the human melanoma cell lines. GPNMB plays a key role in the anti-inflammatory and antioxidative functions as well as osteoblast differentiation, cancer progression, and tissue regeneration. Recently, GPNMB was used as an anti-aging vaccine for mice. The present study aimed to investigate the potential of biofluid GPNMB as an aging biomarker in humans using serum and urine samples from an aging Chinese population. Methods: We analyzed RNA-sequencing data (GSE132040) from 17 murine organs across different ages to assess the gene expression of potential ageing biomarkers. Spearman's correlation coefficients were used to evaluate the relationship between gene expression and age. Meanwhile, a cross-sectional population study was conducted, which included 473 participants (aged 25–91 years), a representative subset of participants from the Peng Zu Study on Healthy Ageing in China (Peng Zu Cohort). Biofluid GPNMB levels were measured by ELISA. The associations of serum and urine GPNMB levels with various clinical and anthropometrical indices were assessed using ANOVA, Kruskal-Wallis H test, and univariate and multivariate linear regression analyses. Results: In mice, the Gpnmb mRNA expression levels showed a significant positive association with age in multiple organs in mice (P

This article proposes a singularity-free fixed-time neuro-adaptive control strategy for robot manipulators, with the goal of addressing trajectory-tracking challenges presented by model uncertainties, external disturbances, and input saturation. To mitigate the impact of input saturation, an auxiliary system is introduced. Combining the backstepping technique, a fixed-time neuro-adaptive controller is designed to ensure that tracking errors converge within a small region around the origin within a fixed time, with the upper bound of convergence time being independent of initial conditions. Notably, the direct avoidance of singularity is achieved by constructing quadratic-fraction functions in both the virtual controller and the actual controller, eliminating the need for filters or piecewise continuous functions. This simplifies and streamlines the stability analysis process. To validate the effectiveness of this strategy, numerical simulations are conducted.

BackgroundPlatelet hyperreactivity is a risk factor for thrombosis in elderly patients with cardiovascular diseases. However, the mechanism of platelet hyperactivation has not been elucidated. This study aims to investigate alterations in the proteomes of platelets and their correlation with platelet hyperreactivity among elderly individuals.MethodsThis study included 10 young (28.1 ± 1.9 years), 10 middle-aged (60.4 ± 2.2 years), and 10 old (74.2 ± 3.0 years) subjects. Washed platelets were used in the present study. Platelet samples were analysed by using data-independent acquisition (DIA) quantitative mass spectrometry (MS).ResultsThe results showed that the platelet proteomic profile exhibited high similarity between the young and middle-aged groups. However, there were significant differences in protein expression profiles between the old group and the young group. By exploring the dynamic changes in the platelet proteome with ageing, clusters of proteins that changed significantly with ageing were selected for further investigation. These clusters were related to the initial triggering of complement, phagosome and haemostasis based on enrichment analysis. We found that platelet degranulation was the major characteristic of the differentially expressed proteins between the old and young populations. Moreover, complement activation, the calcium signalling pathway and the nuclear factor-κB (NF-κB) signalling pathway were enriched in differentially expressed proteins.ConclusionsThe present study showed that there are obvious differences in the protein profiles of the elderly compared with young and middle-aged populations. The results provide novel evidence showing changes in platelet hyperactivity and susceptibility to thrombosis in the elderly population.

Amino acid variants in protein may result in deleterious effects on enzymatic activity. In this study we investigate the DNA variants on activity of CYP2B6 gene in a Chinese Han population for potential use in precision medicine. All exons in CYP2B6 gene from 1483 Chinese Han adults (Zhejiang province) were sequenced using Sanger sequencing. The effects of nonsynonymous variants on recombinant protein catalytic activity were investigated in vitro with Sf12 system. The haplotype of novel nonsynonymous variants with other single nucleotide variants in the same allele was determined using Nanopore sequencing. Of 38 alleles listed on the Pharmacogene Variation Consortium, we detected 7 previously reported alleles and 18 novel variants, of which 11 nonsynonymous variants showed lower catalytic activity (0.00–0.60) on bupropion compared to CYP2B6*1. Further, these 11 novel star-alleles (CYP2B6*39–49) were assigned by the Pharmacogene Variation Consortium, which may be valuable for pharmacogenetic research and personalized medicine.

AbstractContext Poziotinib and vonoprazan are two drugs mainly metabolized by CYP3A4. However, the drug-drug interaction between them is unknown.Objective To study the interaction mechanism and pharmacokinetics of poziotinib on vonoprazan.Materials and methods In vitro experiments were performed with rat liver microsomes (RLMs) and the contents of vonoprazan and its metabolite were then determined with UPLC-MS/MS after incubation of RLMs with vonoprazan and gradient concentrations of poziotinib. For the in vivo experiment, rats in the poziotinib treated group were given 5 mg/kg poziotinib by gavage once daily for 7 days, and the control group was only given 0.5% CMC-Na. On Day 8, tail venous blood was collected at different time points after the gavage administration of 10 mg/kg vonoprazan, and used for the quantification of vonoprazan and its metabolite. DAS and SPSS software were used for the pharmacokinetic and statistical analyses.Results In vitro experimental data indicated that poziotinib inhibited the metabolism of vonoprazan (IC50 = 10.6 μM) in a mixed model of noncompetitive and uncompetitive inhibition. The inhibitory constant Ki was 0.574 μM and the binding constant αKi was 2.77 μM. In vivo experiments revealed that the AUC(0-T) (15.05 vs. 90.95 μg/mL·h) and AUC(0-∞) (15.05 vs. 91.99 μg/mL·h) of vonoprazan increased significantly with poziotinib pretreatment. The MRT(0-∞) of vonoprazan increased from 2.29 to 5.51 h, while the CLz/F value decreased from 162.67 to 25.84 L/kg·h after pretreatment with poziotinib.Conclusions Poziotinib could significantly inhibit the metabolism of vonoprazan and more care may be taken when co-administered in the clinic.

Resistance to temozolomide (TMZ), the frontline chemotherapeutic agent for glioblastoma (GBM), has emerged as a formidable obstacle, underscoring the imperative to identify alternative therapeutic strategies to improve patient outcomes. In this study, we comprehensively evaluated a novel agent, O6-methyl-2′-deoxyguanosine-5′-triphosphate (O6-methyl-dGTP) for its anti-GBM activity both in vitro and in vivo. Notably, O6-methyl-dGTP exhibited pronounced cytotoxicity against GBM cells, including those resistant to TMZ and overexpressing O6-methylguanine-DNA methyltransferase (MGMT). Mechanistic investigations revealed that O6-methyl-dGTP could be incorporated into genomic DNA, disrupting nucleotide pools balance, and inducing replication stress, resulting in S-phase arrest and DNA damage. The compound exerted its anti-tumor properties through the activation of AIF-mediated apoptosis and the parthanatos pathway. In vivo studies using U251 and Ln229 cell xenografts supported the robust tumor-inhibitory capacity of O6-methyl-dGTP. In an orthotopic transplantation model with U87MG cells, O6-methyl-dGTP showcased marginally superior tumor-suppressive activity compared to TMZ. In summary, our research, for the first time, underscores the potential of O6-methyl-dGTP as an effective candidate against GBM, laying a robust scientific groundwork for its potential clinical adoption in GBM treatment regimens.

Genetic polymorphism of the cytochrome P450 (CYP) gene can significantly influence the metabolism of endogenous and xenobiotic compounds. However, few studies have focused on the polymorphism of CYP2J2 and its impact on drug catalytic activity, especially in the Chinese Han population. In this study, we sequenced the promoter and exon regions of CYP2J2 in 1,163 unrelated healthy Chinese Han individuals using the multiplex PCR amplicon sequencing method. Then, the catalytic activities of the detected CYP2J2 variants were evaluated after recombinant expression in S. cerevisiae microsomes. As a result, CYP2J2*7, CYP2J2*8, 13 variations in the promoter region and 15 CYP2J2 nonsynonymous variants were detected, of which V15A, G24R, V68A, L166F and A391T were novel missense variations. Immunoblotting results showed that 11 of 15 CYP2J2 variants exhibited lower protein expression than wild-type CYP2J2.1. In vitro functional analysis results revealed that the amino acid changes of 14 variants could significantly influence the drug metabolic activity of CYP2J2 toward ebastine or terfenadine. Specifically, 4 variants with relatively higher allele frequencies, CYP2J2.8, 173_173del, K267fs and R446W, exhibited extremely low protein expression and defective catalytic activities for both substrates. Our results indicated that a high genetic polymorphism of CYP2J2 could be detected in the Chinese Han population, and most genetic variations in CYP2J2 could influence the expression and catalytic activity of CYP2J2. Our data significantly enrich the knowledge of genetic polymorphisms in CYP2J2 and provide new theoretical information for corresponding individualized medication in Chinese and other Asian populations.

Objective(s): This study aims to evaluate the catalytic activities of 31 CYP2C19 alleles and their effects on the metabolism of tapentadol in vitro. Materials and Methods: Insect microsomes expressing the CYP2C19 alleles were incubated with 50–1250 μM tapentadol for 40 min at 37 °C and terminated by cooling to -80 °C, immediately. Tapentadol and N-desmethyl tapentadol were analyzed by a UPLC-MS/MS system. The kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl tapentadol were determined. Results: As a result, the intrinsic clearance (Vmax/Km) values of most variants were significantly altered, while CYP2C19.3 and 35FS had no detectable enzyme activity. Only one variant, N277K, showed no significant difference from CYP2C19.1B. Two variants CYP2C19.29 and L16F displayed markedly increased intrinsic clearance values of 302.22% and 199.97%, respectively; whereas 24 variants exhibited significantly decreased relative clearance ranging from 0.32% to 79.15% of CYP2C19.1B. Especially, CYP2C19.2G, 2H, R124Q, and R261W exhibited a drastic decrease in clearance (>80%) compared with wild-type CYP2C19.1B. Conclusion: As the first study of all aforementioned alleles for tapentadol metabolism, the comprehensive data in vitro may provide novel insights into the allele-specific and substrate-specific activity of CYP2C19.

Abstract Background The major histocompatibility complex (MHC) in humans includes three classical class I loci (A, B, and C), which are important biomarkers for the transplantation of organs and hematopoietic stem cells. In the MHC, polymorphism is known to be extremely high while interlocus recombination is rare. We report a rare interlocus recombination between HLA-A and HLA-H, which was analyzed using next generation sequencing and nanopore sequencing. Methods In the sample, the genotypes of HLA-A, B, C, DRB1, and DQB1 were firstly determined using the methods of sequence-specific primer, sequence-specific oligonucleotide, Sanger’s sequencing, and NGS; however, HLA-A could not be phased. Nanopore sequencing was finally utilized to distinguish the sequence of the novel allele. Results Finally, the novel HLA-A*11:335 allele was identified as an interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles; this was mainly achieved by nanopore sequencing. Conclusions The identification of the interlocus recombination indicated that nanopore sequencing can be helpful in the characterization of novel alleles with complex rearrangements. Interlocus recombination has been identified as one of the mechanisms involved in the generation of novel HLA alleles.

BackgroundDiabetes mellitus (DM), a metabolic disease that has attracted significant research and clinical attention over the years, can affect the eye structure and induce cataract in patients diagnosed with DM. Recent studies have indicated the relationship between glycoprotein non-metastatic melanoma protein B (GPNMB) and DM and DM-related renal dysfunction. However, the role of circulating GPNMB in DM-associated cataract is still unknown. In this study, we explored the potential of serum GPNMB as a biomarker for DM and DM-associated cataract.MethodsA total of 406 subjects were enrolled, including 60 and 346 subjects with and without DM, respectively. The presence of cataract was evaluated and serum GPNMB levels were measured using a commercial enzyme-linked immunosorbent assay kit.ResultsSerum GPNMB levels were higher in diabetic individuals and subjects with cataract than in those without DM or cataract. Subjects in the highest GPNMB tertile group were more likely to have metabolic disorder, cataract, and DM. Analysis performed in subjects with DM elucidated the correlation between serum GPNMB levels and cataract. Receiver operating characteristic (ROC) curve analysis also indicated that GPNMB could be used to diagnose DM and cataract. Multivariable logistic regression analysis illustrated that GPNMB levels were independently associated with DM and cataract. DM was also found to be an independent risk factor for cataract. Further surveys revealed the combination of serum GPNMB levels and presence of DM was associated with a more precise identification of cataract than either factor alone.ConclusionsIncreased circulating GPNMB levels are associated with DM and cataract and can be used as a biomarker of DM-associated cataract.

Cytochrome 2C9 (CYP2C9), one of the most important drug metabolic enzymes in the human hepatic P450 superfamily, is required for the metabolism of 15% of clinical drugs. Similar to other CYP2C family members, CYP2C9 gene has a high genetic polymorphism which can cause significant racial and inter-individual differences in drug metabolic activity. To better understand the genetic distribution pattern of CYP2C9 in the Chinese Han population, 931 individuals were recruited and used for the genotyping in this study. As a result, seven synonymous and 14 non-synonymous variations were identified, of which 4 missense variants were designated as new alleles CYP2C9*72, *73, *74 and *75, resulting in the amino acid substitutions of A149V, R150C, Q214H and N418T, respectively. When expressed in insect cell microsomes, all four variants exhibited comparable protein expression levels to that of the wild-type CYP2C9 enzyme. However, drug metabolic activity analysis revealed that these variants exhibited significantly decreased catalytic activities toward three CYP2C9 specific probe drugs, as compared with that of the wild-type enzyme. These data indicate that the amino acid substitution in newly designated variants can cause reduced function of the enzyme and its clinical significance still needs further investigation in the future.

Infigratinib (INF) is a fibroblast growth factor receptor (FGFR)-specific tyrosine kinase inhibitor for the therapy of advanced cholangiocarcinoma. However, CYP3A4 polymorphisms and CYP3A4 inducers or inhibitors might affect the pharmacokinetics of INF. Clinical evaluation of drug-drug interactions and adverse effects in these patients was necessary with reference to INF levels in vivo. The presently conducted study optimized a reproducible and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analytic technique, which was validated and applied to determine INF concentrations in Sprague-Dawley (SD) rat plasma and pharmacokinetic studies. Protein was precipitated by adding acetonitrile to plasma samples, followed by gradient elution on a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) for complete chromatographic separation of the analyte and derazantinib (used as internal standard, IS). A gradient elution of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.30 mL/min was applied as the mobile phase for this analysis. The ion transitions of INF and IS were m/z 599.88 → 313.10 and m/z 468.96 → 382.00 during UPLC-MS/MS detection, respectively, by multiple reaction monitoring (MRM). The methodological validation demonstrated that INF presented high linearity over the concentration of 2–600 ng/mL. The lower limit of quantification (LLOQ) for this experiment was 2 ng/mL, of which the precisions and accuracies were within the permissible levels. Inter-day and intra-day precisions were demonstrated to be within reasonable limits, which were within 15%, and the accuracies were determined to be between 2.2% and 11.4%. Moreover, the values of recovery, stability and matrix effect of INF were within the limits of acceptability. The pharmacokinetics of INF in SD rats was investigated by gavage administration of 10 mg/kg INF, followed by the application of the developed UPLC-MS/MS analytical method to detect the content in plasma and derive the main pharmacokinetic parameters.

This study aimed to explore the effect of baicalein on the pharmacokinetics of cilostazol (CLZ) and its two metabolites 3,4-dehydro cilostazol (3,4-CLZ) and 4′-trans-hydroxy cilostazol (4′-CLZ) in rats using a newly established ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. Ticagrelor was used as an internal standard (IS), then cilostazol and its two metabolites were separated by means of a UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using gradient elution method with 0.4 ml/min of flow rate. Acetonitrile as organic phase and water with 0.1% formic acid as aqueous phase constructed the mobile phase. Selective reaction monitoring (SRM) mode and positive ion mode were preferentially chosen to detect the analytes. Twelve SD rats were divided into two groups (n = 6) when CLZ was administered orally (10 mg/kg) with or without oral baicalein (80 mg/kg). The selectivity, linearity, recovery, accuracy, precision, matrix effect and stability of UPLC-MS/MS assay were satisfied with the standards of United States Food and Drug Administration guidelines. In control group, AUC0-∞ and Cmax of CLZ were 2,169.5 ± 363.1 ng/ml*h and 258.9 ± 82.6 ng/ml, respectively. The corresponding results were 3,767.6 ± 1,049.8 ng/ml*h and 308.6 ± 87.9 ng/ml for 3, 4-CLZ, 728.8 ± 189.9 ng/ml*h and 100.3 ± 51.3 ng/ml for 4′-CLZ, respectively. After combination with baicalein, AUC0-∞ and Cmax of CLZ were 1.48, 1.38 times higher than the controls. Additionally, AUC0-∞ and Cmax were separately decreased by 36.12 and 19.54% for 3,4-CLZ, 13.11 and 44.37% for 4′-CLZ. Baicalein obviously alters the pharmacokinetic parameters of CLZ, 3,4-CLZ and 4′-CLZ in rats. These results suggested that there was a potential drug-drug interaction between baicalein and CLZ. Therefore, it must raise the awareness when concomitant use of CLZ with baicalein, the dosage regimen of CLZ should be taken into consideration, if this result is confirmed in clinical studies.

Objective: The purpose of this study was to compare the levels of 8-oxoG and 8-oxodG in urine of patients with cervical carcinoma and healthy women to evaluate their influences on cervical carcinoma. Methods: In this study, urine samples were collected from 70 patients with cervical carcinoma, 24 patients with one-year follow-up, and 100 healthy women. The contents of 8-oxodG and 8-oxoG in urine were assayed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Results: The levels of 8-oxoG and 8-oxodG were higher in patients with cervical carcinoma (P < 0.000), while AUC of 8-oxoG and 8-oxodG was higher than 0.7. Specifically, the high-risk HPV (HR-HPV) positive group had higher 8-oxoG levels (P < 0.000), but there was no difference in 8-oxodG levels. Yet, 8-oxoG level was associated with lymphatic metastasis, lymph-vascular space infiltration (LVSI) and stromal infiltration, while 8-oxodG level was affected by the differentiation degree and stromal infiltration. According to statistics, the distinct cut-off index of lymphatic metastasis was 7.282 nmol/mmol creatinine. After operation, the concentrations of 8-oxoG and 8-oxodG dropped significantly (8-oxoG P < 0.000, 8-oxodG P = 0.004). Except for chemotherapy group, the urinary 8-oxoG dose of all treatment groups and 8-oxodG dose of chemo-radiotherapy group declined obviously. Conclusions: 8-oxoG may be a potential biomarker for cervical carcinoma.

Abstract Background AUF1 is one of the AU‐rich binding proteins, which promotes rapid ARE‐mRNA degradation. Recently, it has been reported that AUF1 is involved in regulating the antioxidant system because of its capacity to bind specifically to RNA containing oxidized bases and degrade oxidized RNA. Many antioxidant proteins have been reported to be overexpressed in colorectal cancer (CRC), however, the role of AUF1 in the progression of CRC has not been explored. Methods The expression level of AUF1 protein in human CRC cell lines and CRC tissues was detected by western blotting and immunohistochemistry (IHC. The effects of AUF1 knockdown on CRC cell proliferation, migration, invasion and changes in the signaling pathways were evaluated using a cell counting kit‐8 (CCK‐8), Transwell assays and western blotting. Subcutaneous xenograft tumor model was employed to further substantiate the role of AUF1 in CRC. Results AUF1 protein was upregulated in CRC tissues and CRC cells, and high expression of AUF1 was significantly associated with advanced AJCC stage (P = .001), lymph node metastasis (P = .007), distant metastasis (P = .038) and differentiation (P = .009) of CRC specimens. CRC patients with the high expression of AUF1 had an extremely poor prognosis. The knockdown of AUF1 suppressed CRC cell line proliferation, migration and invasion, inhibited CRC cells tumorigenesis and growth in nude mice, and reduced phosphorylated‐ERK1/2 and phosphorylated AKT in CRC cells. Conclusion Our findings demonstrate that AUF1 is probably involved in the progression of CRC via the activation of the ERK1/2 and AKT pathways. AU‐rich RNA‐binding factor 1 could be used as a novel prognostic biomarker and a potential therapeutic target for CRC.

Pneumatic muscle is a relatively new pneumatic component in some precise and flexible control systems and such systems are needed to achieve a higher controlling performance. However, the existence of practical uncertainties will make it hard to achieve a higher control accuracy and better performance. In this paper, the adaptive control problem for the pneumatic muscle joint system with input saturation, external disturbance and modeling errors is investigated. The auxiliary signals r1, r2 and function ψsgn as an approximation of sign(·) function is introduced to deal with the unknown saturation and uncertainties caused by modeling errors. Then an adaptive control scheme is proposed by using these auxiliary signals and functions. Finally, simulation studies are used to verify the effectiveness of the proposed control scheme.

In this paper, the position tracking control for tank gun control systems with periodic reference signal is studied. On the basis of corresponding system modeling, a novel repetitive controller is developed by using Lyapunov synthesis. During the controller design, signal replacement mechanism is used to deal with the nonparametric uncertainties under Lipschitz-like continuous condition, and repetitive learning laws are developed to estimate the unknown periodic parameters. Meanwhile, robust learning approach is used to compensate the sum of random disturbances, whose upper bound is estimated according repetitive learning mechanism. Hyperbolic tangent function, rather than sign function, is applied to design a robust feedback term to release the occurrence of chattering phenomenon. Numerical simulations demonstrate the effectiveness of the proposed repetitive control scheme.

IntroductionHealthy aging is described as a process of developing and maintaining intrinsic abilities, including physical and cognitive functions. Although oxidative stress is a common mechanism shared by loss of muscle strength and dementia, its relationship with decreased physical performance and cognitive impairment remains unclear. We aimed to investigate the role of urinary 8-oxo-7, 8-dihydroguanosine (8-oxoGsn), a biomarker of oxidative damage to RNA, in physical and cognitive decline.MethodsThe study followed a cross-sectional design and recruited 40–94-year-old inhabitants of Beijing, China (471 men and 881 women). The physical performance of the participants was assessed using handgrip strength, walking speed, and the repeated chair stand test. The cognitive function was assessed using the Montreal Cognitive Assessment (MoCA) 5-min protocol. Urinary 8-oxoGsn levels were measured for all participants.ResultsParticipants with high urinary 8-oxoGsn levels were more likely to have low grip strength, slow walking speed, poor performance in the repeated chair stand test, and low scores on the MoCA 5-min protocol (odds ratio [OR] 3.43, 95% confidence interval [CI]: 1.52–7.76; OR 1.71, 95% CI: 1.16–2.53; OR 2.06, 95% CI: 0.92–4.63; OR 1.75, 95% CI: 1.18–2.58), after adjusting for age, sex, smoking habits, alcohol consumption, hypertension, diabetes, cerebro-cardiovascular disease, and chronic kidney disease.ConclusionElevated levels of oxidative stress are independently associated with cognitive and physical impairment. Thus, these results can help in the early identification and development of strategies for the prevention and treatment of intrinsic capacity decline.

Oxidative RNA damage has been found to be associated with age-related diseases and 8-oxo-7,8-dihydroguanosine (8-oxoGsn) is a typical marker of oxidative modification of RNA. Urine tests are a feasible non-invasive diagnostic modality. The present study aimed to assess whether the measurement of urinary 8-oxoGsn could represent a potential early maker in mild cognitive impairment (MCI) of frail patients with cardiovascular disease (CVD). In this cross-sectional study performed in China from September 2018 to February 2019. Urinary 8-oxoGsn was measured in frail (Fried phenotype: 3–5) in patients with CVD and was adjusted by urinary creatinine (Cre) levels. Cognitive function was assessed by the Chinese version of the Mini-Mental State Examination (MMSE) and participants were classified into non-MCI (≥24) and MCI (

Adaptive backstepping control is an effective method for servo system driven by twin motors with unknown actuator failures, however, problems such as explosion of complexity make it can not be used in the actual control systems. In this paper, an algorithm of adaptive dynamic surface control is proposed for servo system driven by twin motors with unknown actuator failures, which can eliminate the expansion of the differential term and simplify calculations in the controller design. In addition, all parameters and disturbances in the system model are unknown, which require to be identified online. The simulation results also provided in order to show the effectiveness of the algorithm.

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

Abstract Cytochrome P450 2C9 (CYP2C9) is one of the most important drugs metabolizing enzymes and accounts for the metabolism of about 13%–17% of clinical drugs. Like other members in CYP2 family, CYP2C9 gene exhibits great genetic polymorphism among different races and individuals. CYP2C9*18 is one CYP2C9 allelic variant identified in a Southeast Asian population and is estimated to cause the amino acid substitutions of I359L and D397A in CYP2C9 enzyme simultaneously. Limited by the low expression level in bacteria and COS‐7 cells, no valuable enzyme kinetics have been reported on this CYP2C9 variant. In this study, the baculovirus‐based system was used for the high expression of recombinant CYP2C9 s in insect cells. As a result, together with I359L substitution, D397A could significantly decrease the protein expression of CYP2C9.18 in insect cells, although substitution of D397A alone had no effect on the expression of CYP2C9 in vitro. As compared with that of wild‐type enzyme, both CYP2C9.18 variant and D397A variant could decrease more than 80% of the catalytic activity of CYP2C9 enzyme toward three probe substrates, suggesting that caution should be exercised when patients carrying CYP2C9*18 taking medicines metabolized by CYP2C9 enzyme with a narrow therapeutic window.

NUDIX hydrolase type 5 (NUDT5) is a kind of ADP-ribose pyrophosphatase and nucleotide metabolizing enzyme in cell metabolism. Previous studies have shown NUDT5 expression affected chromosome remodeling, involved in cell adhesion, cancer stem cell maintenance and epithelial to mesenchyme transition in breast cancer cells. Nevertheless, the role of NUDT5 in breast cancer progression and prognosis has not yet been systematically studied. This study explored the association of NUDT5 with the tumor development and poor prognosis in patients with breast cancer. Our results show that the levels of NUDT5 were upregulated in breast cancer cell lines and breast tumor tissues, and the expression of NUDT5 in breast tumor tissues increased significantly when compared with adjacent non-tumorous tissues by immunohistochemical staining of tissue microarrays. Breast cancer patients with high NUDT5 expression had a worse prognosis than those with low expression of NUDT5. In addition, the knockdown of NUDT5 suppressed breast cancer cell lines proliferation, migration and invasion, and dramatically inhibited the AKT phosphorylation at Thr308 and expression of Cyclin D1. The opposite effects were observed in vitro following NUDT5 rescue. Our findings indicated that the high expression of NUDT5 is probably involved in the poor prognosis of breast cancer via the activation of the AKT / Cyclin D pathways, which could be a prognostic factor and potential target in the diagnosis and treatment of breast cancer.

Background MTH1 and NUDT5 effectively degrade nucleotides containing 8-oxoguanine. MTH1 and NUDT5 have been linked to the malignancy of multiple cancers. However, their functions in tumor growth and metastasis in esophageal squamous carcinoma (ESCC) remain obscure. Our present study aims to explore their prognostic value in ESCC and investigate their function in MTH1 or NUDT5-knockout tumor cells. Methods MTH1 and NUDT5 protein expression in ESCC adjacent normal tissues and tumor tissues was examined by immunohistochemistry staining. Kaplan–Meier curves were used to assess the association between their expression and overall survival (OS) in ESCC patients. Univariate and Multivariate Cox regression analyses were generated to determine the correlation between these protein expression and OS of ESCC patients. Protein expression in ESCC cell lines were measured by Western blotting. To explore the potential effects of the MTH1 and NUDT5 protein in ESCC, cell models with MTH1 or NUDT5 depletion were established. CCK-8, cell cycle, Western blotting, migration and invasion assays were performed. Results Our present study demonstrated that the levels of MTH1 and NUDT5 were upregulated in ESCC cell lines and ESCC tissues, the expression of MTH1 and NUDT5 in ESCC tissues was significantly higher than in adjacent non-tumorous, and higher levels of MTH1 and NUDT5 predicted a worse prognosis in patients with ESCC. MTH1 and NUDT5 are novel biomarkers of the progression of ESCC and a poor prognosis. We also found for the first time that the high expression of NUDT5 independently predicted lower OS in patients with ESCC (hazard ratio (HR) 1.751; 95% confidence interval (CI) [1.056–2.903]; p = 0.030). In addition, the depletion of MTH1 and NUDT5 strongly suppressed the proliferation of ESCC cells and significantly delayed the G1 phase of the cell cycle. Furthermore, we found that MTH1 and NUDT5 silencing inhibited epithelial–mesenchymal transition mainly by the MAPK/MEK/ERK dependent pathway, which in turn significantly decreased the cell migration and invasion of ESCC cells. Our results suggested that the overexpression of MTH1 and NUDT5 is probably involved in the tumor development and poor prognosis of ESCC.

This paper investigates the controller design problem for mean-square asymptotical synchronization of discrete-time neural networks with time-varying delay. We proposed the design method of synchronization controller, which considered the nonlinearity of controller input. Based on the designed controller, a delay-dependent synchronization criterion is proposed and formulated in the form of linear matrix inequalities (LMIs) by applying the Lyapunov function method. The result is extended to the delayed discrete-time neural network with uncertainty. Two numerical examples are presented to illustrate the effectiveness of the proposed method.

Abstract Lesinurad is an oral inhibitor of urate-anion exchanger transporter 1 and has been approved by the US Food and Drug Administration for combination therapy with a xanthine oxidase inhibitor for the treatment of hyperuricemia associated with refractory gout. In the present study, a sensitive and specific ultra high-performance liquid chromatography with tandem mass spectrometry assay was established and verified for the determination of lesinurad in rat plasma and was described in details for the first time. Chromatographic separation of lesinurad and diazepam (internal standard, IS) was performed on a Rapid Resolution HT C18 column (3.0 × 100 mm, 1.8 µm) using methanol–water (70:30, v/v) as the mobile phase at a flow rate of 0.3 mL/min. Lesinurad and IS were extracted from plasma by liquid–liquid extraction using ethyl acetate. The mass spectrometric detection was carried out using an electrospray ionization source in positive mode. Multiple reaction monitoring was used for quantification of the precursor to product ion at m/z 405.6 → 220.9 for lesinurad and m/z 285.1 → 192.8 for IS. The assay was well validated for selectivity, accuracy, precision, recovery, linearity, matrix effects, and stability. The verified method was applied to obtain the pharmacokinetic parameters and concentration–time profiles for lesinurad after oral/intravenous administration in rats. The study might provide an important reference and a necessary complement for the qualitative and quantitative evaluation of lesinurad.

Background: Cytochrome P450 proteins (CYP 450) is the most important enzyme system of drug phase I metabolism in liver. In previous reports, reduced efficiency or increased risk of adverse events can be affected by primary sequence mutation in CYP450. Aim: To investigate the effect of gene polymorphism on the metabolism of ketamine in vitro, including the new alleles: 2C9*58, *59 and *60. Method: Incubation system which was contained insect microsome, b5, NADPH and 1M PBS incubated 10 μM–1000 μM ketamine in 37 °C for 40 min concentration of norketamine was analyzed by ultra-performance liquid chromatography–tandem mass spectrometry system (UPLC-MS/MS). Result: Catalytic activity of thirty-eight CYP2C9 alleles on ketamine metabolism to norketamine was surveyed. Compared with 2C9*1, three alleles (2C9*40, *49 and *51) was demonstrated dramatically increased intrinsic clearance (1.2-fold–3.75-fold); four subtypes (2C9*27, *31, *41 and *56) exhibited no significantly change on enzyme activity. The resting 31 alleles expressed different degrees of reduction compared with wild type. Conclusion: The result of research warns that attention should be more paid on individual who carry on the special 2C9 alleles under the situation of administrating ketamine.

Cytochrome P450 3A4 (CYP3A4) enzyme activity is known to show considerable ethnic heterogeneity and inter-individual differences, affecting the outcome of drug treatment. CYP3A4 genetic polymorphisms are believed to be one of the important causes, leading to inter-individual variability in drug metabolism. Quinine is an antipyretic drug with antimalarial properties that is metabolized primarily by CYP3A4. Quinine 3-hydroxylation has been proven as a biomarker reaction for evaluating CYP3A4 ability. Quinine has frequent adverse effects and there are distinct inter-individual differences in quinine sensitivity. The open reading frame for 30 CYP3A4 allelic variants were constructed from wild-type CYP3A4*1A by an overlap extension polymerase chain reaction. Recombinant CYP3A4 variants were expressed using baculovirus-insect cell expression system, and their catalytic activities towards quinine hydroxylation were determined and evaluated. Of the 30 CYP3A4 allelic variants, 23 variants exhibited significantly reduced intrinsic clearance towards quinine, 2 variants showed increased intrinsic clearance for quinine, 2 variants possessed no significant differences towards quinine, compared with CYP3A4*1A, and 3 variants had no detected expression and enzyme activity. Our assessment on the enzymatic activities of CYP3A4 variants towards quinine may contribute to laying an experimental foundation for further clinical studies so as to accelerate the process of determining the associations between genetic variations and clinical phenotypes.

Background: A molecular biomarker of physiologic age, as opposed to chronologic age, is needed in clinical medicine. 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGsn) and 8-oxo-7, 8-dihydroguanosine (8-oxoGsn) are two promising aging biomarkers.Methods: A total of 1,228 healthy Chinese residents (613 males and 615 females) 2–90 years of age were randomly selected. Spot urine samples were collected, and the concentrations of 8-oxodGsn and 8-oxoGsn were measured using ultra-high-performance liquid chromatography with a triple quadrupole mass spectrometer (UPLC-MS/MS). Method validation, including accuracy, precision, linearity and quantification limit, was performed. The relationship between oxidized guanosine and age/gender was evaluated.Results: 8-oxodGsn and 8-oxoGsn were eluted at 1.61 and 1.30 min, respectively. The calibration curve was linear in the range of 0.2–500 ng/ml for both analytes. The lowest limit of quantification (LLOQ) was 0.2 ng/ml for 8-oxodGsn and 0.1 ng/ml for 8-oxoGsn. There was an age-dependent increase in the biomarkers from the 21- to 30-year-old group to the 81- to 90-year-old group in both genders. In the subjects older than 61 years of age, the levels of 8-oxodGsn as well as 8-oxoGsn in urine were much higher in females than in males. The content of 8-oxoGsn correlated more closely with age and was higher (approximately 2-fold) than that of 8-oxodGsn for a given individual.Conclusions: 8-oxodGsn and 8-oxoGsn can be easily measured by UPLC-MS/MS. Urinary 8-oxoGsn may be a potential biomarker to determine a person's physiologic age and identify individuals at high risk of developing age-associated disease.