Evaluation of Calu-3 cell lines as an in vitro model to study the inhalation toxicity of flavoring extracts.

This study aimed to evaluate the characteristics of Calu-3 cells as a model to examine the toxicological responses of inhalable substances. Calu-3 cells were grown to the confluence at an air-liquid interface (ALI) using a Transwell® permeable support system. The ALI resulted in biomimetic native bronchial epithelium displaying pseudostratified columnar epithelium with more microvilli and secretory vesicles. We further characterized and optimized the Calu-3 cell line model using ALI culturing conditions, immunolabeling of protein expression, ultrastructural analysis using scanning electron microscopy (SEM), and transepithelial electrical resistance (TEER) measurements, and then screened for the cytotoxicity of tobacco flavoring extracts. Calu-3 cells displayed dose-dependent responses when treated with the flavoring extract. Within 8-10 days, cell monolayers developed TEER ≥1000 Ω·cm2. During this time, Calu-3 cells exposed to flavoring extracts X01 and X06 exhibited a loss of cellular integrity and decreased ZO-1 and E-cadherin protein expression. In conclusion, we investigated the Calu-3 cell line culture conditions, culture time, and barrier integrity and tested the effect of six new synthetic tobacco flavoring extracts. Our data demonstrate that the Calu-3 human bronchial epithelial cell monolayer system is a potential in vitro model to assess the inhalation toxicity of inhalable substances.

Nutrigenomics reveals potential genetic underpinning of diverse taste preference of Chinese men.

BACKGROUND: Taste preference varies geographically in China. However, studies on Chinese people's taste preference in different regions of China are limited, and are lack of research on the mechanism of differences in taste preference, especially in genetics. OBJECTIVE: This study aims to investigate the characteristics of taste preference of Chinese men, and estimate whether diverse taste preference in Chinese have genetic underpinning. METHODS: We conducted a questionnaire survey on taste preferences on 1076 males from 10 regions of China, and collected another 1427 males from the same regions which genotyped by microarray. We compared the correlation between different taste preference, and evaluated the correlation between the mutation frequency of inhouse database and different taste preference. The putative taste-preference-related genes were further utilized to estimate the candidate relationship on gene and gene network in different taste preference. RESULTS: There was a correlation between different taste preferences in Chinese men. We found 31 SNPs associated with 6 kind of taste preferences. These SNPs located within or nearby 36 genes, and the tastes associated with 4 of these genes (TRPV1, AGT, ASIC2 and GLP1R) are consistent with the previous studies. Moreover, in different tastes which were suggested to be associated with each other, some putative related genes were the same or in the same gene network, such as pathways related with blood pressure, response to stimulus and nervous system. CONCLUSIONS: This study indicates that the diverse taste preference of Chinese men may have genetic underpinning.

Clinical benefit of COX-2 inhibitors in the adjuvant chemotherapy of advanced non-small cell lung cancer: A systematic review and meta-analysis.

BACKGROUND: Lung cancer is a major cause of death among patients, and non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers in many countries. AIM: To evaluate the clinical benefit (CB) of COX-2 inhibitors in patients with advanced NSCLC using systematic review. METHODS: We searched the six electronic databases up until December 9, 2019 for studies that examined the efficacy and safety of the addition of COX-2 inhibitors to chemotherapy for NSCLC. Overall survival (OS), progression free survival (PFS), 1-year survival rate (SR), overall response rate (ORR), CB, complete response (CR), partial response (PR), stable disease (SD), and toxicities were measured with more than one outcome as their endpoints. Fixed and random effects models were used to calculate risk estimates in a meta-analysis. Potential publication bias was calculated using Egger's linear regression test. Data analysis was performed using R software. RESULTS: The COX-2 inhibitors combined with chemotherapy were not found to be more effective than chemotherapy alone in OS, progression free survival, 1-year SR, CB, CR, and SD. However, there was a difference in overall response rate for patients with advanced NSCLC. In a subgroup analysis, significantly increased ORR results were found for celecoxib, rofecoxib, first-line treatment, and PR. For adverse events, the increase in COX-2 inhibitor was positively correlated with the increase in grade 3 and 4 toxicity of leukopenia, thrombocytopenia, and cardiovascular events. CONCLUSION: COX-2 inhibitor combined with chemotherapy increased the total effective rate of advanced NSCLC with the possible increased risk of blood toxicity and cardiovascular events and had no effect on survival index.

Genome-Wide Association Study of Smoking Behavior Traits in a Chinese Han Population.

Tobacco use is one of the leading causes of preventable disease worldwide. Genetic studies have elucidated numerous smoking-associated risk loci in American and European populations. However, genetic determinants for cigarette smoking in Chinese populations are under investigated. In this study, a whole-genome sequencing (WGS)-based genome-wide association study (GWAS) was performed in a Chinese Han population comprising 620 smokers and 564 nonsmokers. Thirteen single-nucleotide polymorphisms (SNPs) of the raftlin lipid linker 1 (RFTN1) gene achieved genome-wide significance levels (P < 5 x 10-8) for smoking initiation. The rs139753473 from RFTN1 and six other suggestively significant loci from CUB and sushi multiple domains 1 (CSMD1) gene were also associated with cigarettes per day (CPD) in an independent Chinese sample consisting of 1,329 subjects (805 smokers and 524 nonsmokers). When treating males separately, associations between smoking initiation and PCAT5/ANKRD30A, two genes involved in cancer development, were identified and replicated. Within RFTN1, two haplotypes (i.e., C-A-C-G and A-G-T-C) formed by rs796812630-rs796584733-rs796349027-rs879511366 and three haplotypes (i.e., T-T-C-C-C, T-T-A-T-T, and C-A-A-T-T) formed by rs879401109-rs879453873-rs75180423-rs541378415-rs796757175 were strongly associated with smoking initiation. In addition, we also revealed two haplotypes (i.e., C-A-G-G and T-C-T-T derived from rs4875371-rs4875372-rs17070935-rs11991366) in the CSMD1 gene showing a significant association with smoking initiation. Further bioinformatics functional assessment suggested that RFTN1 may participate in smoking behavior through modulating immune responses or interactions with the glucocorticoid receptor alpha and the androgen receptor. Together, our results may help understand the mechanisms underlying smoking behavior in the Chinese Han population.

Clinical outcomes of botulinum toxin A management for neurogenic detrusor overactivity: meta-analysis.

The aim of this work was to evaluate the efficacy and safety of botulinum toxin A (BTX-A) treatment in patients with neurogenic detrusor overactivity. PUBMED, EMBASE, and Cochrane Library were identified on 13 May 2017 to identify relevant randomized controlled trials. All data obtained were analyzed using Stata 12.0. Five randomized controlled trials were included in this study. Compared to placebo, the BTX-A groups had significantly fewer urinary incontinence (UI) episodes per day and per week (BTX-A with 300 U for frequency of UI per day at week 2, mean difference (MD): -1.13, 95% confidence interval (CI): -1.89 to -0.37; 200 U; BTX-A with 300 U for frequency of UI per week at week 6, MD: -11.42, 95% CI: -13.91 to -8.93; BTX-A with 200 U for frequency of UI per week at week 6, MD: -10.72, 95% CI: -13.40 to -8.04), increased in maximum cystometric capacity at week 6 (BTX-A with 300 U, MD: 154.88, 95% CI: 133.92-175.84; BTX-A with 200 U, MD: 141.30, 95% CI: 121.28-161.33), decreased maximum detrusor pressure at week 6 (BTX-A with 300 U, MD: -31.72, 95% CI: -37.69 to -25.75; BTX-A with 200 U, MD: -33.47, 95% CI: -39.20 to -27.73). For adverse effects, BTX-A was often associated with more complications and urinary tract infections (BTX-A with 300 U: relative risk (RR):1.42, 95% CI: 1.15-1.76; BTX-A with 200 U: RR: 1.42, 95% CI: 1.11-1.82). This meta-analysis suggests that treatment with BTX-A is effective and safe for neurogenic detrusor overactivity, and recommends using BTX-A with 300 U or with 200 U, as suitable dosage.

Meta-Analysis of Randomized Controlled Trials Using Botulinum Toxin A at Different Dosages for Urinary Incontinence in Patients With Overactive Bladder.

Background: Urinary incontinence (UI) is a common and refractory complication for patients with neurogenic detrusor overactivity (NDO) or idiopathic overactive bladder (IOAB). Objectives: To evaluate the effect of Botulinum toxin A (BTX-A) based on different dosages strategy for UI. Method: The MEDLINE, Ovid EMbase, The Cochrane Central Register of Controlled Trials (CENTRAL), China National Knowledge Internet (CNKI), and WanFang database were searched for relevant published randomized controlled trials (RCTs) between 1969 to September 31, 2018. All database were searched to identify relevant randomized controlled trials (RCTs) that investigated the clinical benefit of BTX-A for management of UI in patients with NDO and IOAB. Results: This meta-analysis involved 19 original studies. The BTX-A was superior to placebo in reducing episodes of UI for NDO patients in all subgroups of different dosages for different durations, and also reduced maximum detrusor pressure in all kinds of 200U and 300U at 6 weeks. However, it increased post void residual in different dosages of 200U at 2 weeks. For IOAB patients, compared to placebo, BTX-A increased detrusor compliance for different dosages of 200U and 300U at 12 and 36 weeks, but it increased risk of urinary tract infections at other dosages. Conclusions: This meta-analysis indicated that BTX-A 200U and 300U are more effective than placebo in the treatment of NDO, with minimal, local, and manageable adverse events. Furthermore, BTX-A 300U and 200U could also improve detrusor compliance of IOAB. However, more RCTs would still be necessary to explore the effect of BTX-A on management of UI in NDO and IOAB patients.

Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry.

More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants.

Discovery and Structural Optimization of N5-Substituted 6,7-Dioxo-6,7-dihydropteridines as Potent and Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors against L858R/T790M Resistance Mutation.

EGFR-targeted inhibitors (gefitinib and erlotinib) provided an effective strategy for the treatment of non-small-cell lung cancer. However, the EGFR T790M secondary mutation has become a leading cause of clinically acquired resistance to these agents. Herein, on the basis of the previously reported irreversible EGFR inhibitor (compound 9), we present a structure-based design approach, which is rationalized via analyzing its binding model and comparing the differences of gatekeeper pocket between the T790M mutant and wild-type (WT) EGFR kinases. Guided by these results, a novel 6,7-dioxo-6,7-dihydropteridine scaffold was discovered and hydrophobic modifications at N5-position were conducted to strengthen nonpolar contacts and improve mutant selectivity over EGFR(WT). Finally, the most representative compound 17d was identified. This work demonstrates the power of structure-based strategy in discovering lead compounds and provides molecular insights into the selectivity of EGFR(L858R/T790M) over EGFR(WT), which may play an important role in designing new classes of mutant-selective EGFR inhibitors.

Comparative study of the contents of analogues of aristolochic acid in two kinds of Aristolochiae Fructus by high-performance liquid chromatography.

Aristolochiae Fructus ("Madouling") is derived from the fruits of Aristolochia contorta and A. debilis (Aristolochiaceae). These two species contain potentially nephrotoxic constituents, but are officially used in China. Distinction of constituents and toxicity between these two species remains unclear. A high-performance liquid chromatography method was developed and validated for the simultaneous determination of seven analogues of aristolochic acid (aristolochic acids I, II, IIIa, IVa and VIIa), as well as aristololactams I and II in Aristolochiae Fructus. Chromatographic separation was achieved on a Zorbax SB-C(18) column with a gradient mobile phase comprising acetonitrile and 1 % acetic acid-30 mM triethylamine (20:1, v/v) buffer. Analytes were detected with a diode array detector at 250 and 260 nm. The contents of seven constituents in samples (11 batches of A. contorta fruits, 15 batches of A. debilis fruits and 33 commercial samples of Madouling) were determined. The content of aristolochic acid IVa was higher than that of aristolochic acid VIIa in A. contorta fruits, whereas the opposite was true in A. debilis fruits. This feature can be used to distinguish the two species from each other and identify the resource plant of Madouling. Through a morphological method and a newly found principle based on the ratio AA-IVa/AA-VIIa, we found that the 33 commercial samples collected from 12 provinces in China were all derived from the fruits of A. contorta.

[Relationship between early coagulability parameters at admission and outcome in patients with severe heatstroke].

OBJECTIVE: To discuss the relationship between early coagulability parameters at admission in patients with severe heatstroke and their outcome. METHODS: The data from 176 patients with severe heatstroke admitted to Guangzhou General Hospital of Guangzhou Military Command from January 1st, 2002 to August 31st, 2013 were retrospectively analyzed. The patients were divided into survival group (n=150) and non-survival group (n=26) according to the outcome. The incipient values of coagulability function indexes within 24 hours after admission were collected, and prothrombin time (PT), activated partial thromboplastin time (APTT) and platelet count (PLT) were compared between two groups to assess the statistically significant indexes for the analysis of the relationship between coagulability parameters and outcome with receiver operator characteristic curve (ROC curve). RESULTS: Compared with those in survival group, PT and APTT were significantly prolonged in non-survival group [PT: 34.0 (18.8, 45.6) s vs. 18.4 (13.8, 18.0) s, Z=-6.09, P=0.000; APTT: 79.7 (41.0, 91.2) s vs. 60.8 (33.4, 41.0) s, Z=-5.08, P=0.000]. The PLT counts were significantly lower in the non-survival group than those in survival group [ 60.8(4.7, 95.3) × 109/L vs. 128.4(79.8, 180.8) × 109/L, Z=-4.34, P=0.000]. ROC curve analysis showed that the area under ROC curve (AUC) for PT in predicting the death of patients with severe heatstroke was 0.874, with standard error of 0.028 and 95% confidence interval (95%CI) of 0.819-0.927 (P=0.000). The best cut-off was 18.5 s, with sensitivity of 76.9% and specificity of 20.0%. AUC for APTT in predicting the death of patients with severe heatstroke was 0.812, with standard error of 0.047 and 95%CI of 0.740-0.903 (P=0.000). The best cut-off was 46.55 s, with sensitivity of 69.2% and specificity of 14.0%. AUC for PLT in predicting the death of patients with severe heatstroke was 0.767, with standard error of 0.040 and 95%CI of 0.688-0.845 (P=0.000). The best cut-off was 86.5 × 109/L, with sensitivity of 68.0% and specificity of 36.8%. CONCLUSIONS: Early prolonged PT and APTT and reduced PLT count are associated with increased risk of death, and it can predict a poor outcome in patients with severe heatstroke.

[Chemical constituent from fruit of Aristolochia contorta].

OBJECTIVE: To study the chemical constituents of the fruit of Aristolochia contorta. METHOD: The compounds were isolated by chromatographic techniques and crystalization, the structures were elucidated by spectrum analysis. RESULT: Fifteen compounds were isolated from the dry fruit of A. contorta, which were six aristolochic acids: aristolochic acid I, aristolochic acid III a, aristolochic acid IVa, aristolochic acid II, aristolochic acid III and aristolochic acid VIIa. Three aristolactams: aristololactam I, aristololactam II and aristololactam IIIa. Three phenolic acids syringic acid, vanillic acid and p-coumaric acid. Three other type compounds: pentacosane acid, beta-sitosterol and daucossterol. CONCLUSION: Aristolochic acid III, aristolochic acid VIIa, aristololactam IIIa, and penfacosane acid were isolated from A. contorta for the first time, and compounds 4-13 were isolated from the furit of A. contorta for the first time.

Simultaneous determination of five aristolochic acids and two aristololactams in Aristolochia plants by high-performance liquid chromatography.

An HPLC method was developed for the simultaneous determination of five aristolochic acids (AAs) and two aristololactams (ALs) in the following six Chinese drugs derived from Aristolochia species. Samples were analyzed on a C(18) column with acetonitrile and 3.7 mm phosphoric acid buffer gradient elution, detected at 260 nm. Assay was linear over the range (microg/mL) 0.386-38.6 for aristolochic acid Va, 0.632-63.2 for aristolochic acid IVa, 0.200-20.0 for 9-hydroxy aristolochic acid I, 0.352-35.2 for aristololactam II, 0.296-29.6 for aristolochic acid II, 0.274-27.4 for aristololactam I and 3.12-312 for aristolochic acid I. Average recoveries (%) of samples were 102.0, 95.9, 99.2, 102.2, 97.2, 97.1 and 97.8 for these seven constituents, respectively. The detection limit and retention time for the seven constituents ranged from 10.0 to 15.8 ng/mL and from 12 to 21 min. As a result of drug determination, contents (in mg/g) were as follows: AA-I, 0.69-1.77; AA-II, 0.02-0.18; 9-OH AA-I, 0.04-0.12; AA-IVa, 0.76-3.36; AA-Va, 0.04-0.31; AL-I, 0.07-0.36; and AL-II, 0.01-0.09 in Madouling; AA-I, 0.03-0.41; AA-II, 0.01-0.11; 9-OH AA-I, 0.00-0.60; AA-IVa, 0.00-0.77; AA-Va, 0.00-0.14; and AL-I, 0.00-0.04 in Tianxianteng; AA-I, 1.19-4.71; and AA-II, 0.24-1.69 in Qingmuxiang; AA-I, 2.79-5.48; AA-II, 1.06-1.86; 9-OH AA-I, 0.01-0.09; AA-IVa, 0.38-0.69; AA-Va, 0.00-0.61; AL-I, 0.00-0.02; and AL-II, 0.00-0.02 in Bei-madouling-gen; AA-I, 0.64-4.23; AA-II, 0.06-0.40; and AA-IVa, 0.08-0.25; in Guangfangji; and AA-I, 1.88-9.72; AA-II, 0.26-1.88; and AA-IVa, 0.09-0.52 in Guanmutong. The other constituents were not detected in Tianxianteng, Qingmuxiang, Guangfangji and Guanmutong.