Determination of misoprostol free acid in human breast milk and serum by gas chromatography/negative ion chemical ionization tandem mass spectrometry

To study an expected transition of misoprostol from human blood into breast milk, a novel method for the determination of its active metabolite misoprostol acid (MPA) was developed. MPA was determined in serum and breast milk samples by an isotope dilution assay using gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS). After addition of (15S)-15-methylprostaglandin E(2) (15-methyl-PGE(2)) as an internal standard, MPA was extracted from both matrices using a reversed-phase cartridge. The prostanoids were derivatized with O-2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride (PFBHA) and 2,3,4,5,6-pentafluorobenzyl bromide (PFBB) to the pentafluorobenzyl oxime (PFBO)-pentafluorobenzyl ester (PFB) derivatives. The sample was subjected to thin-layer chromatography with ethyl acetate-hexane (1 : 1 (v/v)) as the developing solvent. The corresponding zone was extracted. After derivatization to the trimethylsilyl ether, MPA was determined by GC/NICI-MS/MS using the [molecule (M) - pentafluorobenzyl (PFB)](-) ([P](-)) ions as precursor in the negative ion chemical ionization mode. The product ions used for quantification were [P - 2TMSOH - C(6)F(5)CH(2)OH](-) (MPA) and [P - 2TMSOH - C(6)F(5)CH(2)OH - CO(2)](-)(15-methyl-PGE(2)), respectively. The limit of quantification for MPA was approximately 1 pg ml(-1) in breast milk and serum samples. The correlation coefficients of the calibration curves for MPA were r > 0.997 in the 0.5-2000 pg ml(-1) range for both tested matrices.     

Long-term exposure to CdTe quantum dots causes functional impairments in live cells

Several studies suggested that the cytotoxic effects of quantum dots (QDs) may be mediated by cadmium ions (Cd2+) released from the QDs cores. The objective of this work was to assess the intracellular Cd2+ concentration in human breast cancer MCF-7 cells treated with cadmium telluride (CdTe) and core/shell cadmium selenide/zinc sulfide (CdSe/ZnS) nanoparticles capped with mercaptopropionic acid (MPA), cysteamine (Cys), or N-acetylcysteine (NAC) conjugated to cysteamine. The Cd2+ concentration determined by a Cd2+-specific cellular assay was below the assay detection limit (<5 nM) in cells treated with CdSe/ZnS QDs, while in cells incubated with CdTe QDs, it ranged from approximately 30 to 150 nM, depending on the capping molecule. A cell viability assay revealed that CdSe/ZnS QDs were nontoxic, whereas the CdTe QDs were cytotoxic. However, for the various CdTe QD samples, there was no dose-dependent correlation between cell viability and intracellular [Cd2+], implying that their cytotoxicity cannot be attributed solely to the toxic effect of free Cd2+. Confocal laser scanning microscopy of CdTe QDs-treated cells imaged with organelle-specific dyes revealed significant lysosomal damage attributable to the presence of Cd2+ and of reactive oxygen species (ROS), which can be formed via Cd2+-specific cellular pathways and/or via CdTe-triggered photoxidative processes involving singlet oxygen or electron transfer from excited QDs to oxygen. In summary, CdTe QDs induce cell death via mechanisms involving both Cd2+ and ROS accompanied by lysosomal enlargement and intracellular redistribution.     

HPLC-UV assay for monitoring total and unbound mycophenolic acid concentrations in children

A simple, accurate and sensitive HPLC method was developed for measuring total and unbound mycophenolic acid (MPA) in human plasma. Total MPA was extracted by protein precipitation and ultrafiltration was used to assess unbound MPA concentrations. The supernatant (20 microL) or ultrafiltrate (100 microL) was injected onto a C(18) HPLC column with a mobile phase of 0.05 m sodium phosphate buffer (pH 2.31)-acetonitrile (55:45, v/v for total MPA; 50:50 for unbound MPA) with UV detection at 254 nm. The extraction recovery was over 93% and reproducible. The assay was linear over the concentration range of 0.07-50 mg/L for total MPA and 4-1500 microg/L for unbound MPA. Intra- and inter-day assay reproducibility was less than 10%. Detection limits were 0.04 mg/L and 2 microg/L for total and unbound MPA, respectively. The assay utility was established in samples collected from five paediatric bone marrow transplant recipients who were receiving intravenous doses of mycophenolate mofetil. In these patients MPA concentrations ranged from 0.07 to 7.83 mg/L and unbound drug concentrations ranged from 2.1 to 107.5 microg/L. This method can be effectively applied to MPA pharmacokinetics in paediatric patients.     

Specific and sensitive determination of medroxyprogesterone acetate in human serum by gas chromatography-mass spectrometry

The mass fragmentographic determination of medroxyprogesterone acetate (MPA) in serum, using as internal standard medroxyprogesterone propionate (MPP) synthesized from MPA, is described. After addition of MPP, the sera are extracted on Sep-Pak C18 cartridges and MPA and MPP are detected as their respective 3-enol trifluoroacyl esters. Serum samples from 84 patients with breast cancer, daily receiving MPA orally, were determined showing a large variation in MPA concentrations (4-349 ng/ml). Our proposed gas chromatographic-mass spectrometric (GC-MS) method, which can be considered as a reference, was compared with a radioimmunoassay (RIA) method showing a correlation coefficient of 0.73 (n = 69; p much less than 0.001). The assay was also used to determine sequential serum levels of patients receiving a single oral dose of MPA. With only minor adjustments, the GC-MS method allows the determination of serum concentrations of related steroids such as megestrol acetate and cyproterone acetate.     

Polyphenol Oxidase as a Promising Alternative Therapeutic Agent for Cancer Therapy

Cancers have always been the most difficult to fight, the treatment of cancer is still not considered. Thus, exploring new anticancer drugs is still imminent. Traditional Chinese medicine has played an important role in the treatment of cancer. Polyphenol oxidase (PPO) extracted from Edible mushroom has many related reports on its characteristics, but its role in cancer treatment is still unclear. This study aims to investigate the effects of PPO extracted from Edible mushroom on the proliferation, migration, invasion, and apoptosis of cancer cells in vitro and explore the therapeutic effects of PPO on tumors in vivo. A cell counting kit-8 (CCK8) assay was used to detect the effect of PPO on the proliferation of cancer cells. The effect of PPO on cancer cell migration ability was detected by scratch test. The effect of PPO on the invasion ability of cancer cells was detected by a transwell assay. The effect of PPO on the apoptosis of cancer cells was detected by flow cytometry. Female BALB/c mice (18–25 g, 6–8 weeks) were used for in vivo experiments. The experiments were divided into control group, model group, low-dose group (25 mg/kg), and high-dose group (50 mg/kg). In vitro, PPO extracted from Edible mushroom significantly inhibited the proliferation, migration, and invasion capability of breast cancer cell 4T1, lung cancer cell A549, and prostate cancer cell C4-2, and significantly promoted the apoptosis of 4T1, A549, and C4-2. In vivo experiments showed PPO inhibitory effect on tumor growth. Collectively, the edible fungus extract PPO could play an effective role in treating various cancers, and it may potentially be a promising agent for treating cancers.     

Simultaneous quantification of mycophenolic acid and its glucuronide metabolites in human plasma by an UPLC–MS/MS assay

The aim of this study was to develop a reliable UPLC–MS/MS assay for accurate quantification of mycophenolic acid (MPA) and its glucuronide conjugates in human plasma. Plasma proteins were precipitated with acetonitrile and the chromatographic separation was achieved on a C₁₈ column with a gradient elution. The detection was performed by a triple quadrupole mass spectrometer in the positive electrospray ionization and multiple reaction monitoring mode. Linearity of the assay was demonstrated over the range of 20–10,000 ng/mL for MPA and MPA glucuronide (MPAG), and 2–1000 ng/mL for acyl MPA glucuronide in human plasma. The assay was precise and accurate with coefficient of variation and bias <15%. MPA and MPAG were stable at 25 °C up to 1 day in both heparin‐ and EDTA‐treated blood. In heparin‐ and EDTA‐plasma, MPA and MPAG were stable for at least 1 week at 25 and 4 °C, and 1 month at ?20 °C. However, 99% acyl MPA glucuronide degraded in both heparin‐ and EDTA‐blood as well as plasma when stored at room temperature for 1 day. All the analytes remained stable for at least 3 months in acidified EDTA‐plasma at ?80 °C. The assay was successfully applied on patients post hematopoietic stem cell transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

Cytotoxicity and Apoptosis Effects of Curcumin Analogue (2E,6E)-2,6-Bis(2,3-Dimethoxybenzylidine) Cyclohexanone (DMCH) on Human Colon Cancer Cells HT29 and SW620 In Vitro

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. According to the Malaysian National Cancer Registry Report 2012–2016, colorectal cancer was the second most common cancer in Malaysia after breast cancer. Recent treatments for colon cancer cases have caused side effects and recurrence in patients. One of the alternative ways to fight cancer is by using natural products. Curcumin is a compound of the rhizomes of Curcuma longa that possesses a broad range of pharmacological activities. Curcumin has been studied for decades but due to its low bioavailability, its usage as a therapeutic agent has been compromised. This has led to the development of a chemically synthesized curcuminoid analogue, (2E,6E)-2,6-bis(2,3-dimethoxybenzylidine) cyclohexanone (DMCH), to overcome the drawbacks. This study aims to examine the potential of DMCH for cytotoxicity, apoptosis induction, and activation of apoptosis-related proteins on the colon cancer cell lines HT29 and SW620. The cytotoxic activity of DMCH was evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cell viability assay on both of the cell lines, HT29 and SW620. To determine the mode of cell death, an acridine orange/propidium iodide (AO/PI) assay was conducted, followed by Annexin V/FITC, cell cycle analysis, and JC-1 assay using a flow cytometer. A proteome profiler angiogenesis assay was conducted to determine the protein expression. The inhibitory concentration (IC₅₀) of DMCH in SW620 and HT29 was 7.50 ± 1.19 and 9.80 ± 0.55 µg/mL, respectively. The treated cells displayed morphological features characteristic of apoptosis. The flow cytometry analysis confirmed that DMCH induced apoptosis as shown by an increase in the sub-G0/G1 population and an increase in the early apoptosis and late apoptosis populations compared with untreated cells. A higher number of apoptotic cells were observed on treated SW620 cells as compared to HT29 cells. Human apoptosis proteome profiler analysis revealed upregulation of Bax and Bad proteins and downregulation of Livin proteins in both the HT29 and SW620 cell lines. Collectively, DMCH induced cell death via apoptosis, and the effect was more pronounced on SW620 metastatic colon cancer cells, suggesting its potential effects as an antimetastatic agent targeting colon cancer cells.     

Synthesis of chalcones with anticancer activities

Several chalcones were synthesized and their in vitro cytotoxicity against various human cell lines, including human breast adenocarcinoma cell line MCF-7, human lung adenocarcinoma cell line A549, human prostate cancer cell line PC3, human adenocarcinoma cell line HT-29 (colorectal cancer) and human normal liver cell line WRL-68 was evaluated. Most of the compounds being active cytotoxic agents, four of them with minimal IC?? values were chosen and studied in detail with MCF-7 cells. The compounds 1, 5, 23, and 25 were capable in eliciting apoptosis in MCF-7 cells as shown by multiparameter cytotoxicity assay and caspase-3/7, -8, and -9 activities (p < 0.05). The ROS level showed 1.3-fold increase (p < 0.05) at the low concentrations used and thus it was concluded that the compounds increased the ROS level eventually leading to apoptosis in MCF-7 cells through intrinsic as well as extrinsic pathways.     

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Mycophenolic acid (MPA), a frequently used immunosuppressant, exhibits large inter‐patient pharmacokinetic variability. This study (a) developed and validated a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for MPA and metabolites [MPA glucuronide (MPAG) and acyl‐glucuronide (AcMPAG)] in the culture medium of HepaRG cells; and (b) characterized the metabolism interaction between MPA and p‐cresol (a common uremic toxin) in this in vitro model as a potential mechanism of pharmacokinetic variability. Chromatographic separation was achieved with a C₁₈ column (4.6 × 250 mm,5 μm) using a gradient elution with water and methanol (with 0.1% formic acid and 2 mm ammonium acetate). A dual ion source ionization mode with positive multiple reaction monitoring was utilized. Multiple reaction monitoring mass transitions (m/z) were: MPA (320.95 → 207.05), MPAG (514.10 → 303.20) and AcMPAG (514.10 → 207.05). MPA‐d₃ (323.95 → 210.15) and MPAG‐d₃ (517.00 → 306.10) were utilized as internal standards. The calibration curves were linear from 0.00467 to 3.2 μg/mL for MPA/MPAG and from 0.00467 to 0.1 μg/mL for AcMPAG. The assay was validated based on industry standards. p‐Cresol inhibited MPA glucuronidation (IC₅₀ ≈ 55 μm ) and increased MPA concentration (up to >2‐fold) at physiologically relevant substrate‐inhibitor concentrations (n = 3). Our findings suggested that fluctuations in p‐cresol concentrations might be in part responsible for the large pharmacokinetic variability observed for MPA in the clinic.     

Simple high-performance liquid chromatographic method for the determination of medroxyprogesterone acetate in human plasma

The high-performance liquid chromatographic (HPLC) method was developed as a simple, reliable alternative to available methods for measuring plasma concentrations of medroxyprogesterone acetate (MPA). The HPLC method has been successfully automated and is suitable for the rapid, inexpensive analysis of large batches of plasma samples. The best approach involves a solvent extraction followed by HPLC separation and analysis. MPA can be efficiently extracted, at all pH values, by nonpolar solvents. The Spherisorb 5-ODS2 HPLC column provides excellent separation of MPA from endogenous steroids of similar structure and from extraneous plasma blank peaks. A batch of 30-40 samples can be prepared by HPLC analysis in 2-3 hours, with a chromatographic run time of 10 minutes/sample. Calibration curves between 5-250 ng/ml show a good correlation between peak height ratio and MPA concentration, even at low levels. Plasma concentrations of MPA in patients receiving 1 g/day were between 12.6-270 ng/ml in this study, suggesting that the sensitivity of this method, 10 ng/ml, is sufficient for monitoring therapeutic concentrations of MPA. The results show a wide individual variation in plasma concentrations following similar dosing schedules--a finding reported by other workers.     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.     

Synthesis of Novel Methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates and Antitumor Activity Evaluation: Studies In Vitro and In Ovo Grafts of Chick Chorioallantoic Membrane (CAM) with a Triple Negative Breast Cancer Cell Line

A series of novel functionalized methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a–2h were synthesized by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothieno[3,2-b]pyridine-2-carboxylate with (hetero)aryl pinacol boranes, trifluoro potassium boronate salts or boronic acids. Their antitumoral potential was evaluated in two triple negative breast cancer (TNBC) cell lines—MDA-MB-231 and MDA-MB-468, by sulforhodamine B assay. Their effects on the non-tumorigenic MCF-12A cells were also evaluated. The results demonstrated that three compounds caused growth inhibition in both TNBC cell lines, with little or no effect against the non-tumorigenic cells. The most promising compound was further studied concerning possible effects on cell viability (by trypan blue exclusion assay), cell proliferation (by bromodeoxyuridine assay) and cell cycle profile (by flow cytometry). The results demonstrated that the GI₅₀ concentration of compound 2e (13 μM) caused a decreased in MDA-MB-231 cell number, which was correlated with a decreased in the % of proliferating cells. Moreover, this compound increased G0/G1 phase and decreased S phases, when compared to control cells (although was not statistic significant). Interestingly, compound 2e also reduced tumor size using an in ovo CAM (chick chorioallantoic membrane) model. This work highlights the potential antitumor effect of a novel methyl 3-arylthieno[3,2-b]pyridine-2-carboxylate derivative.     

Dioxyxanthones from Polygala hongkongensis and Their Cytotoxicity

Two new dioxyxanthones, polyhongkongenoxanthones A and B(1 and 2) were isolated from the herbs of Polygala hongkongensis, together with six known xanthones. Their structures were elucidated on the basis of chemical and spectroscopic evidence. The isolates were tested for their cytotoxicity against three tumor cell lines(HepG2, GLC-82 and MCF-7, HepG2=human hepatocellular carcinoma cells; GLC-82=human lung carcinoma cells; MCF-7= human breast carcinoma cells) by MTT assay, among which polyhongkongenoxanthone B(2), 1,7-dihydroxy-2,3- methylenedioxyxanthone(3) and 1,7-dihydroxy-3,4,8-trimethoxyxanthone(6) are potential antitumor candidate due to their significant cytotoxic effects on the three cell lines..     

Green biosynthesis,characterization, and cytotoxic effect of magnetic iron nanoparticles using Brassica Oleracea var capitata sub var rubra (red cabbage) aqueous peel extract

The green method of nanoparticle synthesis, which is an environment and living-friendly method, is an updated subject that has appeared as an alternative to conventional methods such as physical and chemical synthesis. In this presented study, the green synthesis of magnetic iron oxide nanoparticles (IONPs) from iron (III) chloride by using Brassica oleracea var. capitata sub.var. rubra aqueous peel extract has been reported. The prepared IONPs were characterized with fourier-transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV-VIS), zeta potential, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). The cytotoxic effects of IONPs on MCF-7 breast cancer cell line were studied by MTT assay, and migrative effect of its were carried out by the wound healing assay. It was found that the mean particle size of IONPs was 675 ± 25 nm, and the polydispersity index was 0.265 PDI. It was also determined that these nanoparticles had an anti-proliferative impact on the MCF-7 breast cancer cell line depending on the dosage. Characterization results support the successful synthesis of nanoparticles, and the dose-dependent cytotoxic effects of nanoparticles on MCF-7 cells also make it a potential chemotherapeutic agent for breast cancer treatment.     

Fully automated microchip system for the detection of quantal exocytosis from single and small ensembles of cells

A lab-on-a-chip device that enables positioning of single or small ensembles of cells on an aperture in close proximity to a mercaptopropionic acid (MPA) modified sensing electrode has been developed and characterized. The microchip was used for the detection of Ca(2+)-dependent quantal catecholamine exocytosis from single as well as small assemblies of rat pheochromocytoma (PC12) cells. The frequency of events increased considerably upon depolarization of the PC12 cell membrane using a high extracelluar concentration of potassium. The number of recorded events could be correlated with the number of cells immobilized on the electrode. Quantal characteristics, such as the number of released molecules per recorded event, are equivalent to data obtained using conventional carbon fiber microelectrodes. The detection sensitivity of the device allows for the detection of less than 10 000 dopamine molecules in a quantal release. The distribution of peak rise-time and full width at half maximum was constant during measurement periods of several minutes demonstrating the stability of the MPA modified surface.     

一种萘酰亚胺衍生物的合成及其体外抗癌活性测定

合成了萘酰亚胺衍生物2-[2-(二甲基氨基)乙基]-6-2-(2-羟乙胺基)乙胺基-1H-苯并异喹啉-1,3(2H)-二酮;利用元素分析、核磁共振谱及质谱分析了其组成和结构;利用MTT法测定了其对人肝癌细胞(HepG2)、人乳腺癌细胞(MDA-MB-231)及人结肠癌细胞(HCT-116)的体外活性.结果表明,标题化合物的体外抗肿瘤活性优于对照品氨萘非特.     

m-THPC-Mediated Photodynamic Therapy (PDT) Does Not Induce Resistance to Chemotherapy, Radiotherapy or PDT on Human Breast Cancer Cells In Vitro

Photodynamic therapy (PDT) uses laser light to activate a photosensitizer that has been absorbed preferentially by cancer cells after systemic administration. A photo-toxic reaction ensues resulting in cell death and tissue necrosis. Some cells, however, may survive PDT. This study was performed to determine if surviving human breast cancer cells (MCF-7) can become resistant to PDT, chemotherapy or radiotherapy. The MCF-7 cells were cultured under standard conditions prior to being exposed to the photosensitizer, 5,10,15,20-meta-tet-ra(hydroxyphenyl)chlorin (zn-THPC), for 24 h and then irradiated with laser light (652 nm). Surviving cells were allowed to regrow by allowing a 2 week interval between each additional PDT. After the third and final treatment, colony formation assays were used to evaluate the sensitivity of cultured cells to ionizing radiation and PDT and the ATP cell viability assay tested in vitro chemosen-sitivity. Flow cytometry was used to analyze the cell cycle. No alterations in the cell cycle were observed after three cycles of PDT with m-THPC. Similar responses to chemotherapy and ionizing radiation were seen in control and treatment groups. The m-THPC-sensitized PDT did not induce resistance to subsequent cycles of PDT, chemo- or radiotherapy. Photodynamic therapy with m-THPC may represent a novel adjunctive treatment of breast cancer that may be combined with surgery, chemotherapy or ionizing radiation.     

细胞代谢组学用于羽扇豆醇干预人乳腺癌细胞MCF-7的机理探究

应用基于气相色谱-质谱联用（GC-MS）的代谢组学方法结合细胞周期实验,研究羽扇豆醇体外抑制人乳腺癌细胞MCF-7增殖的作用机理。代谢组学的研究结果表明：通过正交偏最小方差判别分析（OPLS-DA）可以很好地区分羽扇豆醇作用的MCF-7细胞代谢谱与对照组细胞代谢谱,模型参数为：R²Ycum=0.988,Q²Ycum=0.964。VIP（variable importance in the projection）值大于1的差异代谢物进一步用t检验进行单位分析,选择t<0.05（VIP>1）的代谢物作为羽扇豆醇作用组的生物标志物,得到琥珀酸、磷酸、亮氨酸、异亮氨酸等11种代谢差异物。结合羽扇豆醇将细胞周期抑制在G1期这一现象,推测羽扇豆醇可能是主要抑制了三羧酸循环中的琥珀酰辅酶A的生成和底物磷酸化生成ATP的反应来抑制MCF-7细胞的增殖。本实验从代谢组学角度为乳腺癌抗肿瘤机制提供新的线索。