Long noncoding RNA ARRDC1-AS1 is activated by STAT1 and exerts oncogenic properties by sponging miR-432-5p/PRMT5 axis in glioma.

Dysfunction of long noncoding RNA (lncRNA) is associated with tumorigenesis of various malignancies, including glioma. Previously, lncRNA ARRDC1 antisense RNA 1(ARRDC1-AS1) has been reported to be dysregulated in several tumors. However, the roles of ARRDC1-AS1 in glioma have not been investigated. In this study, we firstly reported that ARRDC1-AS1 expression was distinctly increased in both glioma specimens and cell lines, and high ARRDC1-AS1 expression was associated with advanced clinical progression and poor prognosis of glioma patients. Additionally, STAT1 could activate the transcription of ARRDC1-AS1. Functional studies revealed that knockdown of ARRDC1-AS1 suppressed the proliferation, migration and invasion of glioma cells. Mechanisms exploration indicated ARRDC1-AS1 served as a sponge of miR-432-5p to upregulate PRMT5 expressions. Rescue experiments indicated that knockdown of miR-432-5p reversed the inhibiting effects of ARRDC1-AS1 knockdown on glioma cells. Overall, our findings highlighted the importance of STAT1/ARRDC1-AS1/miR-432-5p/PRMT5 axis in glioma progression and offered novel strategies for glioma treatments.

Antiproliferative Phenothiazine Hybrids as Novel Apoptosis Inducers against MCF-7 Breast Cancer.

We designed a series of novel phenothiazine-1,2,3-triazole hybrids by the molecular hybridization strategy and evaluated their antiproliferative activity against three cancer cell lines (MDA-MB-231, MDA-MB-468 and MCF-7). For the structure-activity relationships, the importance of 1,2,3-triazole and substituents on phenyl ring was explored. Among these phenothiazine-1,2,3-triazole hybrids, compound 9f showed the most potent inhibitory effect against MCF-7 cells, with an IC50 value of 0.8 µM. Importantly, compound 9f could induce apoptosis against MCF-7 cells by regulating apoptosis-related proteins (Bcl-2, Bax, Bad, Parp, and DR5). These potent phenothiazine-1,2,3-triazole hybrids as novel apoptosis inducers might be used as antitumor agents in the future.

Folic acid-conjugated TiO2-doped mesoporous carbonaceous nanocomposites loaded with Mitoxantrone HCl for chemo-photodynamic therapy.

Recently, porous carbons have showed great potential in many areas. In this study, TiO2-doped mesoporous carbonaceous (TiO2@C) nanoparticles were obtained by a simple one-pot hydrothermal treatment, folic acid (FA) was conjugated to TiO2@C through an amide bond, then Mitoxantrone HCl (MTX) was adsorbed onto TiO2@C-FA and a drug delivery system, TiO2@C-FA/MTX was obtained. TiO2@C-FA/MTX showed a much faster MTX release at pH 4.5 than at pH 6.0 and pH 7.4. Furthermore, compared with free MTX, this drug delivery system showed a dose-dependent cytotoxicity by varying the irradiance, and afforded higher antitumor efficacy in cultured PC3 cells in vitro. The ability of TiO2@C-FA/MTX to combine chemotherapy with photodynamic activity enhanced the cancer cell killing effect in vitro, demonstrating that TiO2@C-FA/MTX has a great potential for cancer therapy in the future.

Methylenetetrahydrofolate reductase 677TT genotype may be associated with an increased lung cancer risk in North China: an updated meta-analysis.

BACKGROUND: Although many epidemiology studies have investigated the methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and their associations with lung cancer (LC), definite conclusions cannot be drawn. To clarify the effects of MTHFR polymorphisms on the risk of LC, we performed a meta-analysis in Chinese populations. MATERIAL/METHODS: Related studies were identified from PubMed, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure (CNKI), and Chinese Biology Medicine (CBM) until 16 February 2014. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the associations. RESULTS: A total of 11 studies with 2487 LC cases and 3228 controls were included in this meta-analysis. Overall, no significant association was found between MTHFR C677T polymorphism and LC risk when all studies in Chinese populations were pooled into this meta-analysis. In subgroup analyses stratified by geographical location and source of controls, significantly increased risk was found in North China (T vs. C: OR=1.28, 95% CI: 1.14-1.44; TT vs. CC: OR=1.67, 95% CI: 1.33-2.10; TT + CT vs. CC, OR=1.39, 95% CI=1.15-1.69; TT vs. CC + CT: OR=1.46, 95% CI: 1.03-2.06) and in population-based studies (TT vs. CC: OR=1.37, 95% CI: 1.14-1.65; TT vs. CC + CT: OR=1.25, 95% CI: 1.07-1.45). CONCLUSIONS: This meta-analysis provides evidence that MTHFR C677T polymorphism may contribute to LC development in North China. Studies with larger sample sizes and wider spectrum of populations are warranted to verify this finding.

Preparation and characterization of fullerene (C60) amino acid nanoparticles for liver cancer cell treatment.

The properties of an ideal photosensitizer are water solubility, low cytotoxicity in the dark, high ability to produce reactive oxygen species (ROS). The characteristics of water-soluble fullerene (C60) amino acid nanoparticles as a photosensitizer were evaluated. C60 modified with l-phenylalanine (C60-phe) or glycine (C60-gly) was very efficient to carry out photodynamic activity leading to cleavage of plasmid DNA in vitro. These C60 amino acid nanoparticles were the most active photosensitizer against human Liver cancer cells and induced cancer cells apoptosis after illumination. However, these derivatives exhibited no significant cytotoxicity in dark. It produced diffuse intracellular fluorescence when 2',7'-dichlorfluorescein-diacetate (DCFH-DA) was added as an ROS probe, suggesting phototoxicity of these derivatives related with the generation of intracellular ROS. These findings indicate that these fullerene derivatives may be excellent candidate PDT enhancing agents.

hOGG1: A novel mediator in nitrosamine-induced esophageal tumorigenesis.

BACKGROUND: The effect of human 8-Oxoguanine DNA Glycosylase (hOGG1) on exogenous chemicals in esophageal squamous cell carcinoma (ESCC) remain unclear. The study plans to determine hOGG1 expression levels in ESCC and possible interactions with known environmental risk factors in ESCC. MATERIAL AND METHODS: We analyzed levels of exposure to urinary nitrosamines in volunteers from high and low prevalence areas by GC-MS. And we performed the interaction between hOGG1 gene and nitrosamine disinfection by-products by analyzing hOGG1 gene expression in esophageal tissues. RESULTS: In ESCC, nitrosamine levels were significantly increased and hOGG1 mRNA expression levels were significantly decreased. There was a statistically significant interaction between reduced hOGG1 mRNA levels and non-tap drinking water sources in ESCC. The apparent indirect association between ESCC and NMEA indicated that 33.4% of the association between ESCC and NMEA was mediated by hOGG1. CONCLUSION: In populations which exposed to high levels of environmental pollutants NDMA, low expression of hOGG1 may promote the high incidence of esophageal cancer in Huai'an. hOGG1 may be a novel mediator in nitrosamine-induced esophageal tumorigenesis.

MgZnO p-n heterostructure light-emitting devices.

MgZnO heterostructure light-emitting devices (LEDs) have been fabricated from p-Mg(0.35)Zn(0.65)O/n-Mg(0.20)Zn(0.80)O structures, and the p-type Mg(0.35)Zn(0.65)O film was realized using a lithium-nitrogen codoping method. Obvious ultraviolet emission peaked at around 355 nm dominates the electroluminescence (EL) spectra of the device at room temperature, which comes from the near-band-edge emission of the n-type Mg(0.20)Zn(0.80)O film. This is the first report on MgZnO heterostructured LEDs and the shortest EL emission ever reported in ZnO-based p-n junction LEDs to the best of our knowledge.

Enhanced responsivity of photodetectors realized via impact ionization.

To increase the responsivity is one of the vital issues for a photodetector. By employing ZnO as a representative material of ultraviolet photodetectors and Si as a representative material of visible photodetectors, an impact ionization process, in which additional carriers can be generated in an insulating layer at a relatively large electric field, has been employed to increase the responsivity of a semiconductor photodetector. It is found that the responsivity of the photodetectors can be enhanced by tens of times via this impact ionization process. The results reported in this paper provide a general route to enhance the responsivity of a photodetector, thus may represent a step towards high-performance photodetectors.

Pharmacokinetics of hydroxycamptothecin nanosuspensions in rats.

The purpose of the present study was to investigate the pharmacokinetics of hydroxycamptothecin nanosuspensions after intravenous administration in rats. Hydroxycamptothecin injection was studied parallelly. The results showed that AUC(0 --> infinity), MRT, t1/2(alpha) and t1/2(beta) of hydroxycamptothecin nanosuspensions was significantly higher, while their total body clearance was lower than those of hydroxycamptothecin injections. The results indicate that hydroxycamptothecin nanosuspensions significantly increase hydroxycamptothecin blood concentrations and retention within the systemic circulation.

Preparation and characterization of freeze-dried 2-methoxyestradiol nanoparticle powders.

Poorly water-soluble compounds are difficult to develop as drug products using conventional formulation techniques and are frequently abandoned early in discovery. In the present study, a nanoprecipitation-high-frequency ultrasonication technique was adapted to produce drug nanosuspensions. The formulation of 2-methoxyestradiol (2-ME) as nanosuspension, either in the form of lyophilized powder or granules, was very successful in enhancing dissolution rate, more 45 times than bulk 2-ME being dissolved in the first 10 min. The increase in vitro dissolution rate may favourably affect bioavailability. The nanosuspension produced was then characterized using particle size determination, zeta potential measurement, scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and X-ray analysis. Results showed that freeze-dried nanosuspension composed of amorphous particles with a mean particle size of 244 +/- 10.6 nm (polydispersity index of 0.21 +/- 0.02) was obtained. Physical stability studies showed that 2-ME nanosuspension remained homogeneous with slight increase in mean particle size and polydispersity index over a 3-month period.

Unhealthy Lifestyle Is an Important Risk Factor of Idiopathic BPPV.

Background: Benign paroxysmal positional vertigo (BPPV) is a self-limiting and recurrent disease but the cost is considerable. The number of patients with BPPV increased significantly under the quarantine policy in Hangzhou. The unhealthy lifestyle risk factors of BPPV have not yet been investigated. Thus, the objective is to analyze whether an unhealthy lifestyle is a risk factor of BPPV. Methods: One hundred and sixty three patients with idiopathic BPPV aged 22-87 years (BPPV group), and 89 aged 23-92 years sex-matched control subjects (non-BPPV group) were enrolled in this study. All BPPV patients received a definitive diagnosis which excluded secondary BPPV. Non-BPPV cases excluded BPPV, sudden deafness, Meniere's disease, ear or craniofacial surgery, vestibular neuritis, and head trauma history. We obtained a blood lipids profile, serum uric acid, total bilirubin, and related diagnostic information through the electronic medical record system. To get the time of physical activities and recumbent positions, we asked the patient or their family from February 2020 to June 2020, and the rest of the patient's information was acquired by phone or WeChat. Data Analyses: The t-test or chi-squared test, univariate, and multiple logistic regression analyses were performed for the two groups. For each factor, odds ratios were calculated with 95% confidence intervals (CIs). Moreover, test equality of two or more receiver operating characteristic (ROC) analyses were applied to the physical activities, and recumbent position time; area under curve (AUC) measures were calculated with 95% CIs and compared with each other. Results: The BPPV group had unhealthy lifestyles such as poor physical activities, prolonged recumbent position time, and low rate of calcium or VD supplementation in univariate logistic regression analyses (P < 0.05). Poor physical activities and prolonged recumbent position time were independently associated with BPPV in multiple logistic regression models (OR = 18.92, 95% CI: 6.34-56.43, p = 0.00 and OR = 1.15, 95% CI: 1.01-1.33, p < 0.04). In the comparison of ROC curves of recumbent position time and physical activities in identifying BPPV, AUCs were 0.68 (0.61-0.74), and 0.68 (0.63-0.73), respectively. Conclusion: We conclude that poor physical activities and prolonged recumbent position time may be independent risk factors for BPPV patients, but hypertension, hyperuricemia, hyperlipidemia, hemoglobin, diabetes, serum bilirubin, CHD, and CI, may not be.

An investigation on intestinal absorption of a new anticancer drug, 2-methoxyestradiol.

In this paper, the intestinal absorptive property of a new anticancer drug, 2-methoxyestradiol (2ME2) was investigated by in situ rat intestinal recirculation perfusion experimental techniques. The results indicated that the concentrations of 2ME2 had no influence on absorption rate constant (ka) of 2ME2 and the small intestinal absorption of 2ME2 was a first-order process with passive diffusion mechanism within the concentration tested. 2ME2 was well absorbed at each intestinal segment except duodenum and the best site of intestinal absorption of 2ME2 was the ileum. In addition, the PH of drug perfusate and the concentration of SDS had significant effects on absorption kinetics. Faintly basic environment profitted small intestinal absorption of 2ME2. Tween 80 and SDS could not enhance the intestinal absorption of 2ME2. The above study provided a theoretical foundation for developing effective oral preparations with higher bioavailability to treat cancers. In addition, the improved ligation way for studying the best site of intestinal absorption of 2ME2, should be used extensively in related studies because of decreasing number of experimental rats and saving time.

[Preparation and in vitro and in vivo study of antisense oligodeoxynucleotides-loaded cationic liposomes].

The aim of the paper is to prepare stable antisense oligodeoxynucleotides-loaded cationic liposomes and evaluate the transfection efficiency of asODN to MCF-7 oophoroma cells and study their distribution to different tissues in mice. Antisense oligodeoxynucleotides (asODN)-loaded cationic liposomes were prepared by a thin film-adsorption-lyophilization method which is simple and can overcome crucial pharmaceutical defects (e.g. instability) of liposomes during storage. The morphology was investigated by transmission electron microscope. The size and surface charge of the liposomes were determined by laser particle analyter. The dissociated ligodeoxynucleotides were separated from the liposomes by sephadex column and the entrapment efficiency was determined by using an ultraviolet photometer. Trehalose, mannitol, and glycine were suitable for lyophilization especially trehalose. The resulting liposomes were global microcapsule in a narrow particle size with a mean diameter of 175 nm and 320 nm before and after lyophilization, and a high zeta potentials of +32 mV. The dissociated asODN were separated from the liposomes by sephadex G-50 column and the entrapment coefficient of asODN was 88.4% pre and 83.2% post-lyophilization separately for trehalose. The growth of MCF-7 oophoroma cells were inhibited in vitro obviously (P < 0.05) and transfection efficiency of asODN was 18%, 26%, 44% after 2 h, 4 h and 8 h, respectively. The formulation and method can be used to prepare stable cationic liposomes which can effectively inhibit the growth of MCF-7 oophoroma cells and obtain a high transfection efficiency. This system can improve distribution amount of asODN to tissues especially tumors in mice.

Efficient Red/Near-Infrared-Emissive Carbon Nanodots with Multiphoton Excited Upconversion Fluorescence.

Red/near-infrared (NIR) emissive carbon nanodots (CNDs) with photoluminescence (PL) quantum yield (QY) of 57% are prepared via an in situ solvent-free carbonization strategy for the first time. 1-Photon and 2-photon cellular imaging is demonstrated by using the CNDs as red/NIR fluorescence agent due to the high PL QY and low biotoxicity. Further study shows that the red/NIR CNDs exhibit multiphoton excited (MPE) upconversion fluorescence under excitation of 800-2000 nm, which involves three NIR windows (NIR-I, 650-950 nm; NIR-II, 1100-1350; NIR-III, 1600-1870 nm). 2-Photon, 3-photon, and 4-photon excited fluorescence of the CNDs under excitation of different wavelengths is achieved. This study develops an in situ solvent-free carbonization method for efficient red/NIR emissive CNDs with MPE upconversion fluorescence, which may push forward the application of the CNDs in bioimaging.

[Effect of nanosize delivery system for ASODN against hTERT on the expression of telomerase in the esophageal cancer EC9706 cells].

OBJECTIVE: To investigate the inhibitory effect of nanoparticle-mediated antisense oligodeoxynucleotide (ASODN) of human telomerase reverse transcriptase (hTERT) on telomerase in the esophageal cancer EC9706 cells. METHODS: Line-polyethylenimine (L-PEI) was used to condense ASODN into nanoparticle and to couple NGR peptides into targeting nanoparticle, and the prepared L-PEI/ASODN complexes were transfected into the EC9706 cells. Cellular uptake of L-PEI/ASODN complexes was detected by laser confocal scanning microscopy. MTT assay was used to detect the inhibitory rate of EC9706 cell growth. The level of hTERT mRNA and its protein expression were measured by RT-PCR and immunohistochemistry, respectively. Annexin V FITC/PI double labeling was used to detect cell apoptosis. The distribution of drug in nude mice was observed by laser confocal scanning microscopy, and the growth and morphology of the tumor was examined. RESULTS: The L-PEI-mediated ASODN uptake was enhanced. After transfection, the inhibitory rate of EC9706 cells was time-dependant and there was a significant difference between control cell group and L-PEI/ASODN group (P < 0.05). At 48 h after transfection, the level of hTERT mRNA was decreased significantly compared with that of control cell group (P < 0.05), and the expression of hTERT protein was negative. There was apparent apoptosis in EC9706 cells after transfection with L-PEI/ASODN complexes. For the two NGR/L-PEI/ASODN groups, fluorescence was observed in the liver, kidney, lung and tumor tissues of nude mice, and their uptake intensity was time-dependent. The mean volume of tumors in the two NGR/L-PEI/ASODN groups was significantly smaller than those in blank control group and SODN group (P < 0.05). Apoptotic bodies were detected in the tumors of L-PEI/ASODN group. CONCLUSION: The NGR/L-PEI/ASODN nanoparticles can effectively reach into the human esophageal cancer xenograft and inhibit the tumor growth in nude mice, and this may provide a theoretical and experimental basis for gene therapy for human esophageal squamous cell carcinoma.

Protective effects of paeonol on subacute/chronic brain injury during cerebral ischemia in rats.

Ischemic stroke is a highly complex pathological process that is divided into acute, subacute and chronic phases. Paeonol is a biologically active natural product with a variety of pharmacological effects, including those on neuronal activity. However, the effects of paeonol on subacute/chronic ischemic stroke have remained to be elucidated. The present study was designed to investigate the effects of paeonol against subacute and chronic cerebral ischemic injury and to explore the possible underlying mechanisms. Male adult Sprague Dawley rats were randomly divided into a sham group (treated with saline), a model group [subjected to middle cerebral artery occlusion (MCAO) and treated with saline] and a paeonol-treated group (MCAO + paeonol at 25 mg/kg). Behavioral impairment, infarct volume and ischemic/contralateral hemispheric ratios were assessed at 72 h and at 28 days after MCAO, respectively. Immunofluorescence was employed to determine the neuronal damage and glial responses after MCAO. Compared with the model group, paeonol treatment significantly attenuated behavioral impairment, ischemic infarct volume and moderate cerebral edema in the ischemic brain at 72 h, as well as brain atrophy at 28 days after reperfusion. Furthermore, paeonol treatment ameliorated neuronal damage in the ischemic core and boundary zone regions at 72 h after reperfusion and in the boundary zone at 28 days after reperfusion. In addition, paeonol treatment reduced the proliferation of astrocytes in the boundary zone, and inhibited microglial activation in the ischemic core and boundary zone regions at 72 h and 28 days after reperfusion. These results demonstrated the protective effects of paeonol against subacute/chronic cerebral ischemia, and the mechanism of action may include subacute/chronic microglial activation and astrocyte proliferation.

Neuroprotective effects of Yiqihuoxue calm wind capsule on ischemic stroke in rats.

Stroke remains the third leading cause of death and of adult disability worldwide. Vascular occlusion, followed by ischemic cascade, leads to irreversible tissue injury. Recombinant tissue plasminogen activator is the only FDA approved drug for the current treatment of acute ischemic stroke. However, traditional Chinese medicine has a long history and rich clinical experience in the treatment and rehabilitation of ischemic stroke. Using a classical middle cerebral artery occlusion (MCAO) stroke model, we tested the effectiveness of Yiqihuoxue calm wind (YCW) capsule on neurological function, gross pathology and oxidative stress status in MCAO rats. YCW capsule (3.36 and 6.72 g·kg-1 of crude drug) could significantly lower Longa's score and superoxide dismutase (SOD) level, together with less necrotic cells and infarcted area. In addition to elevated MDA and downregulated iNOS expression, YCW capsule exhibited its neuroprotective effects via free radical scavenging and NO inhibition.

[Inhibition of A549 cells by polybutylcyanoacrylate nanoparticles loaded with antisense oligodeoxynucleotide of hTERT mRNA].

AIM: To investigate the effect of nanoparticles for antisense oligodeoxynucleotide (ASODN) of hTERT mRNA on A549 cells. METHODS: The cationic polybutylcyanoacrylate nanoparticles (NPs) were prepared by an emulsion polymerization process in the presence of DEAE-dextran. Antisense oligodeoxynucleotides were loaded on the particles by adsorption. The cytotoxicity of NPs and proliferation of A549 cells were detected by MTT assay. Intracellular fluorescence intensity after transfecting the 5'-FITC-labelled ASODN (FASODN) and cell cycles were determined by flow cytometry (FCM). Inverse microscope was used to observe the modality of A549 cell transfected by NPs for ASODN. The protein expression of hTERT was measured by immunocytochemistry. RESULTS: The cytotoxicity increased evidently with the increasing concentration of NPs over 2.5 g x L(-1). The intracellular fluorescence in FASODN-NP group was obviously stronger than that in FASODN group (NPs free) after transfection for 24 h (P < 0.01). The inhibitory rate for cell modality change and proliferation after the treatment with ASODN-NP at 72 h reached peak , 62.4% , 44.6% and 36.4% for ASODN1-NP group, ASODN2-NP group and ASODN3-NP group, respectively; The cell cycle in ASODN-NP group varied observably compared with control group and sense oligodeoxynucleotide-nanoparticle (SODN-NP) group and the cell cycle was blocked in G1 phase, the cell number in S phase decreased obviously (P < 0.01); The hTERT protein expression of ASODN-NP group reduced clearly. CONCLUSION: ASODN-NP of hTERT can inhibit the proliferation of A549 cells effectively and cause the change of cell cycle, restraint of protein expression of hTERT and cell viability.

[Modified (Wu's) esophagectomy for a huge thoracic esophageal squamous cell carcinoma 18.3 cm in length].

An esophageal squamous cell carcinoma measuring 18.3 cm in length and 5 cm in diameter was found in the mediastinum of a 53-year man. The patient underwent a modified 3-stage esophagectomy and an esophagogastrostomy at the cervical level (Wu's method). The operation was performed smoothly and the patient recovered uneventfully after the operation. The patient was followed up for 6 months after discharge and reported no difficulties in eating with improved quality of life. This case represents the world's longest esophageal cancer that had been surgically removed. Local advanced esophageal cancer should be removed immediately to prevent potential occurrence of esophageal obstruction, tracheoesophageal fistula or aorto-esophageal fistula.

Influencing Mechanism of the Selenization Temperature and Time on the Power Conversion Efficiency of Cu2ZnSn(S,Se)4-Based Solar Cells.

Cu2ZnSn(S,Se)4 (CZTSSe) films were deposited on the Mo-coated glass substrates, and the CZTSSe-based solar cells were successfully fabricated by a facile solution method and postselenization technique. The influencing mechanisms of the selenization temperature and time on the power conversion efficiency (PCE), short-circuit current density (Jsc), open-circuit voltage (Voc), and fill factor (FF) of the solar cell are systematically investigated by studying the change of the shunt conductance (Gsh), series resistance (Rs), diode ideal factor (n), and reversion saturation current density (J0) with structure and crystal quality of the CZTSSe film and CZTSSe/Mo interface selenized at various temperatures and times. It is found that a Mo(S1-x,Sex)2 (MSSe) layer with hexagonal structure exists at the CZTSSe/Mo interface at the temperature of 500 °C, and its thickness increases with increasing selenization temperature and time. The MSSe has a smaller effect on the Rs, but it has a larger influence on the Gsh, n, and J0. The PCE, Voc, and FF change dominantly with Gsh, n, and J0, while Jsc changes with Rs and Gsh, but not Rs. These results suggest that the effect of the selenization temperature and time on the PCE is dominantly contributed to the change of the CZTSSe/CdS p-n junction and CZTSSe/MSSe interface induced by variation of the quality of the CZTSSe film and thickness of MSSe in the selenization process. By optimizing the selenization temperature and time, the highest PCE of 7.48% is obtained.