Characteristics of pollutant generation from 3D-printed photocured waste combustion.

With the rapid advancement of photopolymerization-based 3D printing technology, the volume of PCW has experienced a sharp increase. The potential environmental ramifications of PCW disposal demand careful consideration, especially given its current practice of being incineration alongside MSW. In this study, the TG-MS/FTIR system was carried out to probe the thermogravimetric characteristics and volatile byproducts during combustion. Various product compositions resulting from different mixing ratios of PCW incineration with MSW were investigated. It was observed that fluorene (C13H10) and triphenylene (C18H12) produced by PCW combustion 0.52 mg/g and 0.43 mg/g respectively, which are twice as abundant as those generated from normal plastic. When PCW incineration along with MSW, compounds such as naphthalene (C10H8), cyclohexane (C6H12), and heptane (C7H16) were generated in concentrations of 1.25 mg/g, 1.05 mg/g, and 0.95 mg/g respectively, which are at least twice as much as with MSW incineration alone. The incineration of PCW with rubber and textiles resulted in the production of 2.34 mg/g to 3.76 mg/g more PAHs compared to PCW combustion alone. The incineration of PCW with paper resulted in the production of 3.12 mg/g to 5.15 mg/g more heptane, nonane, cyclohexane, pyrene, and anthracene than PCW combustion alone. Incineration of PCW with wood proved to be the cleanest method, with product contents primarily below 0.10 mg/g. When incinerated with food residues or normal plastic, most of the product content remained below 0.05 mg/g. Considering the environmental pollution resulting from PCW combustion, the disposal of PCW warrants careful consideration and management.

Growth Performance of and Liver Function in Heat-Stressed Magang Geese Fed the Antioxidant Zinc Ascorbate and Its Potential Mechanism of Action.

The aim of this study was to investigate the in vitro antioxidant activity of zinc ascorbate (AsA-Zn), its effects on the growth performance of and liver function in Magang geese under heat stress, and its potential mechanism. At AsA-Zn concentrations of 7.5, 15, 30, and 60 µmol/L, the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+) radical scavenging rate increased significantly by 120.85%, 53.43%, 36.12%, and 0.99%, respectively, compared with that of ascorbic acid (AsA), indicating that AsA-Zn had better antioxidant performance in vitro. In this study, Magang geese were divided into a control group (basal diet, CON) and experimental groups, who received the basal diet supplemented with 400 mg/kg AsA or 30 (AsA-Zn30), 60 (AsA-Zn60), or 90 (AsA-Zn90) mg/kg AsA-Zn. AsA-Zn supplementation considerably reduced the feed-to-gain ratio, whereas both AsA and AsA-Zn significantly increased the thymus index. Moreover, AsA-Zn supplementation improved serum protein levels, lipid metabolism, liver function, and antioxidant capacity while reducing hepatocyte vacuolar degeneration. Furthermore, supplementation with AsA-Zn60 significantly increased the total antioxidant capacity, glutathione peroxidase activity, and superoxide dismutase activity and decreased the malondialdehyde content in the serum, liver, and hepatic mitochondria (P < 0.05), with more pronounced effects in the AsA-Zn60 group. Moreover, supplementation with ASA-Zn regulated the Nrf 2 signaling pathway and significantly increased the expression of genes encoding antioxidant-related factors in the liver. In conclusion, AsA-Zn has good antioxidant activity, and AsA-Zn supplementation may improve the antioxidant capacity of heat-stressed geese and promote their growth. Supplementation with 30 mg/kg AsA-Zn is recommended.

RTCpredictor: identification of read-through chimeric RNAs from RNA sequencing data.

Read-through chimeric RNAs are being recognized as a means to expand the functional transcriptome and contribute to cancer tumorigenesis when mis-regulated. However, current software tools often fail to predict them. We have developed RTCpredictor, utilizing a fast ripgrep tool to search for all possible exon-exon combinations of parental gene pairs. We also added exonic variants allowing searches containing common SNPs. To our knowledge, it is the first read-through chimeric RNA specific prediction method that also provides breakpoint coordinates. Compared with 10 other popular tools, RTCpredictor achieved high sensitivity on a simulated and three real datasets. In addition, RTCpredictor has less memory requirements and faster execution time, making it ideal for applying on large datasets.

A first comprehensive analysis of Transcribed Ultra Conserved Regions uncovers important regulatory functions of novel non-coding transcripts in gliomas.

Transcribed Ultra-Conserved Regions (TUCRs) represent a severely understudied class of putative non-coding RNAs (ncRNAs) that are 100% conserved across multiple species. We performed the first-ever analysis of TUCRs in glioblastoma (GBM) and low-grade gliomas (LGG). We leveraged large human datasets to identify the genomic locations, chromatin accessibility, transcription, differential expression, correlation with survival, and predicted functions of all 481 TUCRs, and identified TUCRs that are relevant to glioma biology. Of these, we investigated the expression, function, and mechanism of action of the most highly upregulated intergenic TUCR, uc.110, identifying it as a new oncogene. Uc.110 was highly overexpressed in GBM and LGG, where it promoted malignancy and tumor growth. Uc.110 activated the WNT pathway by upregulating the expression of membrane frizzled-related protein (MFRP), by sponging the tumor suppressor microRNA miR-544. This pioneering study shows important roles for TUCRs in gliomas and provides an extensive database and novel methods for future TUCR research.

A first comprehensive analysis of Transcribed Ultra Conserved Regions uncovers important regulatory functions of novel non-coding transcripts in gliomas.

Transcribed Ultra-Conserved Regions (TUCRs) represent a severely understudied class of putative non-coding RNAs (ncRNAs) that are 100% conserved across multiple species. We performed the first-ever analysis of TUCRs in glioblastoma (GBM) and low-grade gliomas (LGG). We leveraged large human datasets to identify the genomic locations, chromatin accessibility, transcription, differential expression, correlation with survival, and predicted functions of all 481 TUCRs, and identified TUCRs that are relevant to glioma biology. Of these, we investigated the expression, function, and mechanism of action of the most highly upregulated intergenic TUCR, uc.110, identifying it as a new oncogene. Uc.110 was highly overexpressed in GBM and LGG, where it promoted malignancy and tumor growth. Uc.110 activated the WNT pathway by upregulating the expression of membrane frizzled-related protein (MFRP), by sponging the tumor suppressor microRNA miR-544. This pioneering study shows important roles for TUCRs in gliomas and provides an extensive database and novel methods for future TUCR research.

The role of microRNAs in brain metastasis.

Brain metastasis (BM) is the most common type of brain tumor and frequently foreshadows disease progression and poor overall survival with patients having a median survival of 6 months. 70,000 new cases of BM are diagnosed each year in the United States (US) and the incidence rate for BM is increasing with improved detection. MicroRNAs (miRNAs) are small non-coding RNAs that serve as critical regulators of gene expression and can act as powerful oncogenes and tumor suppressors. MiRNAs have been heavily implicated in cancer and proposed as biomarkers or therapeutic targets or agents. In this review, we summarize an extensive body of scientific work investigating the role of microRNAs in BM. We discuss miRNA dysregulation, functions, targets, and mechanisms of action in BM and present the current standing of miRNAs as biomarkers and potential therapeutics for BM. We conclude with future directions of miRNA basic and clinical research in BM.

Effect of landfilling time on physico-chemical properties of combustible fractions in excavated waste.

Excavated waste is a byproduct of microbial decomposition and fermentation following landfill disposal. The effective management and utilization of excavated waste offer broad prospects for environmental and resource protection, as well as economic growth. While current research predominantly focuses on plastics in landfills, the physico-chemical properties of excavated waste over extended landfilling time remain unclear. This study aimed to address this gap by excavating waste from a landfill in Tianjin, China, with a maximum landfilling time of 18 years. The findings revealed that, compared to municipal solid waste (MSW), the excavated waste exhibited increased calorific value, ash content, and fixed carbon content after screening the landfill-mined-soil-like-fine fraction. The average calorific value of the excavated waste could reach 57.8 MJ/kg. Additionally, the oxygen content in the excavated combustible waste exceeded that of MSW, increasing from 25.59 % to 34.22 %. This phenomenon is potentially linked to the oxidation of attached soil impurities and waste. The study identified polyethylene (PE), polypropylene (PP), expanded polystyrene (EPS), polyethylene terephthalate (PET), and wood as the primary combustible components. Notably, the excavated waste exhibited a significant decrease in surface gloss, adopting a rough texture with apparent holes, potentially attributed to the acidification and corrosion of organic matter during fermentation. Nevertheless, the breaking of molecular bonds could also contribute to waste fragmentation. Furthermore, an increase in landfilling time resulted in a more pronounced decrease in mechanical properties. For instance, the failure load of PE decreased from 15.61 N to 6.46 N, and PET reduced from 884.83 N to 186.56 N. The chemical composition of excavated waste has changed, with -OH and CO observed in PE with an 18-year landfilling time. In conclusion, these results provide a theoretical foundation for the recycling of excavated waste and contribute to the advancement of waste management and recycling technologies.

[Sources, Disposal Technologies, and Environmental Impact of Photopolymerization-based 3D Printing Plastic Waste].

In the 3D printing industry, photopolymerization-based 3D printing is considered to have the characteristics of high printing accuracy and mature technology. Therefore, it is of wide concern in industrial application and academic research. With the rapid development of photopolymerization-based technology, photopolymerization-based plastic waste will inevitably be produced in the process of product manufacturing and use. This kind of plastic waste is a new type of organic solid waste with an incalculable growth rate, and its impact on the environment is difficult to predict. Based on available research results, the latest research progress of sources, disposal technologies, and environmental impact of photopolymerization-based plastic waste were summarized and analyzed. The results revealed that the photopolymerization-based plastic waste was covalently crosslinked with thermosetting plastic. It had relatively higher activation energy and photo-sensitive chromogenic groups. There were some potential hazards to the environment and biosome caused by the raw material, printing process, and waste disposal process of photopolymerization-based plastic. Therefore, prospects and suggestions were proposed for the possibility of future disposal of photopolymerization-based plastic waste, in order to provide a reference for developing the photopolymerization-based 3D printing industry.

Safety and Pharmacokinetics of PSD502 in Healthy Chinese Male and Female Volunteers: Two Randomized, Double-Blind, Placebo-Controlled, Phase I Trials.

BACKGROUND AND OBJECTIVE: PSD502 is a metered-dose spray for premature ejaculation. The two trials aimed to evaluate the safety and pharmacokinetics of PSD502 in healthy Chinese male and female individuals. METHODS: Two phase I, randomized, double-blind, placebo-controlled trials were conducted in men (Trial 1) and women (Trial 2). The participants were randomized 3:1 to receive PSD502 (7.5 mg of lidocaine and 2.5 mg of prilocaine per spray) or a placebo. For male individuals, a single dose (three sprays) once daily was applied to the glans penis for 21 days except for nine sprays (three doses) on days 7 and 14, 4 h apart for each dose. For female individuals, two sprays were applied to the vagina and one to the cervix once daily for 7 days. The primary endpoint was safety. Pharmacokinetics analysis was also performed. RESULTS: Twenty-four male and 24 female individuals were recruited. Treatment-emergent adverse events occurred in 38.9% (7/18) of male individuals and 66.7% (12/18) of female individuals in the PSD502 group, respectively. Both trials reported 50.0% (3/6) treatment-emergent adverse events for the placebo. No grade ≥ 3 treatment-emergent adverse events, serious adverse events, or treatment-emergent adverse events leading to early withdrawal or discontinuation occurred. After consecutive applications, lidocaine and prilocaine cleared rapidly in both trials. Plasma concentrations exhibited high inter-individual variability. The maximum plasma concentrations of active ingredients were far below the anticipated minimum toxic concentrations. The area under the plasma concentration-time curve of metabolites were ≤ 20% of the parent drugs. No clinically significant accumulations were observed in the two trials. CONCLUSIONS: PSD502 was well tolerated and showed low plasma concentrations in healthy Chinese male and female individuals.

Emerging Strategies for Enhancing Propionate Conversion in Anaerobic Digestion: A Review.

Anaerobic digestion (AD) is a triple-benefit biotechnology for organic waste treatment, renewable production, and carbon emission reduction. In the process of anaerobic digestion, pH, temperature, organic load, ammonia nitrogen, VFAs, and other factors affect fermentation efficiency and stability. The balance between the generation and consumption of volatile fatty acids (VFAs) in the anaerobic digestion process is the key to stable AD operation. However, the accumulation of VFAs frequently occurs, especially propionate, because its oxidation has the highest Gibbs free energy when compared to other VFAs. In order to solve this problem, some strategies, including buffering addition, suspension of feeding, decreased organic loading rate, and so on, have been proposed. Emerging methods, such as bioaugmentation, supplementary trace elements, the addition of electronic receptors, conductive materials, and the degasification of dissolved hydrogen, have been recently researched, presenting promising results. But the efficacy of these methods still requires further studies and tests regarding full-scale application. The main objective of this paper is to provide a comprehensive review of the mechanisms of propionate generation, the metabolic pathways and the influencing factors during the AD process, and the recent literature regarding the experimental research related to the efficacy of various strategies for enhancing propionate biodegradation. In addition, the issues that must be addressed in the future and the focus of future research are identified, and the potential directions for future development are predicted.

Distribution, migration, and removal of N-containing products during polyurethane pyrolysis: A review.

Due to the wide applications of polyurethane (PU), production is constantly increasing, accounting for 8% of produced plastics. PU has been regarded as the 6th most used polymer in the world. Improper disposal of waste PU will result in serious environmental consequences. The pyrolysis of polymers is one of the most commonly used disposal methods, but PU pyrolysis easily produces toxic and harmful nitrogen-containing substances due to its high nitrogen content. This paper reviews the decomposition pathways, kinetic characteristics, and migration of N-element by product distribution during PU pyrolysis. PU ester bonds break to produce isocyanates and alcohols or decarboxylate to produce primary amines, which are then further decomposed to MDI, MAI, and MDA. The nitrogenous products, including NH3, HCN, and benzene derivatives, are released by the breakage of C-C and C-N bonds. The N-element migration mechanism is concluded. Meanwhile, this paper reviews the removal of gaseous pollution from PU pyrolysis and discusses the removal mechanism in depth. Among the catalysts for pollutant removal, CaO has the most superior catalytic performance and can convert fuel-N to N2 by adsorption and dehydrogenation reactions. At the end of the review, new challenges for the utilization and high-quality recycling of PU are presented.

RTCpredictor: Identification of Read-Through Chimeric RNAs from RNA Sequencing Data.

Read-through chimeric RNAs are gaining attention in cancer and other research fields, yet current tools often fail in predicting them. We have thus developed the first read-through chimeric RNA specific prediction method, RTCpredictor, utilizing a fast ripgrep algorithm to search for all possible exon-exon combinations of parental gene pairs. Compared with other ten popular tools, RTCpredictor achieved top performance on both simulated and real datasets. We randomly selected up to 30 candidate read-through chimeras predicted from each software method and experimentally validated a total of 109 read-throughs and on this set, RTCpredictor outperformed all the other methods. In addition, RTCpredictor ( https://github.com/sandybioteck/RTCpredictor ) has less memory requirements and faster execution time.

Pyrolysis properties and kinetics of photocured waste from photopolymerization-based 3D printing: A TG-FTIR/GC-MS study.

The emerging photopolymerization-based 3D printing industry has led to a growing concern for the disposal of photocured waste (PCW), which is inevitably generated during the life cycle of photopolymerization-based 3D printing. In order to shed light on suitable thermochemical treatment and utilization approaches of PCW, this work comprehensively investigated the properties and kinetics during PCW pyrolysis via TG-FTIR/GC-MS analysis. The results demonstrated that the main decomposition of PCW sample happened in the range 320-550 °C with a total weight loss of 93.34 wt%. According to the result of four kinetic models, the activation energy of PCW sample was approximately 228.58-245.05 kJ/mol. Finally, the FTIR and GC-MS results manifested that the main components of volatiles released at different heating rates were the same. The volatiles mainly include (S)-(+)-2-hydroxy-2-phenylprop, benzaldehyde, benzophenone (photo-initiator), benzoic acid, benzoylformic acid etc., which have a multitude of potential applications. However, these volatiles produced by PCW pyrolysis have a certain toxicity and potential hazard. This study demonstrates insightful fundamentals for thermochemical disposal of PCW, which appears to be potentially valuable with the rapid development of the photopolymerization-based 3D printing industry.

Chimeric RNAs Discovered by RNA Sequencing and Their Roles in Cancer and Rare Genetic Diseases.

Chimeric RNAs are transcripts that are generated by gene fusion and intergenic splicing events, thus comprising nucleotide sequences from different parental genes. In the past, Northern blot analysis and RT-PCR were used to detect chimeric RNAs. However, they are low-throughput and can be time-consuming, labor-intensive, and cost-prohibitive. With the development of RNA-seq and transcriptome analyses over the past decade, the number of chimeric RNAs in cancer as well as in rare inherited diseases has dramatically increased. Chimeric RNAs may be potential diagnostic biomarkers when they are specifically expressed in cancerous cells and/or tissues. Some chimeric RNAs can also play a role in cell proliferation and cancer development, acting as tools for cancer prognosis, and revealing new insights into the cell origin of tumors. Due to their abilities to characterize a whole transcriptome with a high sequencing depth and intergenically identify spliced chimeric RNAs produced with the absence of chromosomal rearrangement, RNA sequencing has not only enhanced our ability to diagnose genetic diseases, but also provided us with a deeper understanding of these diseases. Here, we reviewed the mechanisms of chimeric RNA formation and the utility of RNA sequencing for discovering chimeric RNAs in several types of cancer and rare inherited diseases. We also discussed the diagnostic, prognostic, and therapeutic values of chimeric RNAs.

Flue gas torrefaction of municipal solid waste: Fuel properties, combustion characterizations, and nitrogen /sulfur emissions.

Flue gas torrefaction (FGT) was proposed as the pretreatment of the municipal solid waste (MSW) combustion process to improve the fuel properties of MSW and achieve better combustion performance. The optimal FGT parameters were obtained at 300 ℃ and 30 min, with the energy-mass co-benefit index (EMCI) reaching the maximum of 23.38. FGT could significantly increase the heating value and energy density of MSW while reducing the H/C and O/C ratio. Then, the pyrolysis and combustion experiments were performed by tube furnace and TG-MS. The results proved the chemical compositions of MSW were altered, and the heat transfer was enhanced. With FGT, NOx and SO2 emissions could be reduced by 25.7 % and 52.4 %, respectively. This study provides an in-depth understanding of the mechanism of FGT and paves the way for the clean treatment and energy utilization of MSW.

Distribution of Hg during sewage sludge and municipal solid waste Co-pyrolysis: Influence of multiple factors.

Co-pyrolysis is a promising approach to recover energy from sewage sludge (SS) and municipal solid waste (MSW). Hg emission during this process has serious environmental risks. To reduce the environmental impact, orthogonal experiments on the blending ratio, heating rate, pyrolysis temperature, and residence time were conducted during SS and MSW co-pyrolysis. Variance analysis was used to determine the influence and synergetic effects of these factors. Multivariate nonlinear, neural network, random forest, and support vector machine models were used to simulate the Hg distribution based on four parameters, which were later optimized to optimize the Hg fixing ratio in pyrolysis char. The Hg was mainly distributed in the pyrolysis gas and char. The variance analysis results indicate that the blending ratio is the key factor influencing Hg distribution, and there is little synergetic effect among the four factors. Further experiments showed that a blending ratio of 87.5 SS wt% could enhance Hg fixation in char. The neural network model shows the best simulation performance with a mean relative error of 8.92%. The optimal parameters are a heating rate of 7 °C/min, pyrolysis temperature of 300 °C, and residence time of 10 min, resulting in a Hg fixing ratio of 25.68 wt% in pyrolysis char. The simulated Hg fixation characteristics correlate with the experimental results. This study provides insights into Hg distribution under various conditions during co-pyrolysis of SS and MSW. It is hoped that this work can contribute to the control of Hg during the waste treatment and utilization process.

Possible pathways used to predict different stages of lung adenocarcinoma.

We aimed to find some specific pathways that can be used to predict the stage of lung adenocarcinoma.RNA-Seq expression profile data and clinical data of lung adenocarcinoma (stage I [37], stage II 161], stage III [75], and stage IV [45]) were obtained from the TCGA dataset. The differentially expressed genes were merged, correlation coefficient matrix between genes was constructed with correlation analysis, and unsupervised clustering was carried out with hierarchical clustering method. The specific coexpression network in every stage was constructed with cytoscape software. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed with KOBAS database and Fisher exact test. Euclidean distance algorithm was used to calculate total deviation score. The diagnostic model was constructed with SVM algorithm.Eighteen specific genes were obtained by getting intersection of 4 group differentially expressed genes. Ten significantly enriched pathways were obtained. In the distribution map of 10 pathways score in different groups, degrees that sample groups deviated from the normal level were as follows: stage I < stage II < stage III < stage IV. The pathway score of 4 stages exhibited linear change in some pathways, and the score of 1 or 2 stages were significantly different from the rest stages in some pathways. There was significant difference between dead and alive for these pathways except thyroid hormone signaling pathway.Those 10 pathways are associated with the development of lung adenocarcinoma and may be able to predict different stages of it. Furthermore, these pathways except thyroid hormone signaling pathway may be able to predict the prognosis.

Sestrin2 protects the myocardium against radiation-induced damage.

The purpose of this study was to investigate the role of Sestrin2 in response to radiation-induced injury to the heart and on the cardiomyopathy development in the mouse. Mice with genetic deletion of the Sestrin2 (Sestrin2 knockout mice [Sestrin2 KO]) and treatment with irradiation (22 or 15 Gy) were used as independent approaches to determine the role of Sestrin2. Echocardiography (before and after isoproterenol challenge) and left ventricular (LV) catheterization were performed to evaluate changes in LV dimensions and function. Masson's trichrome was used to assess myocardial fibrosis. Immunohistochemistry and Western blot were used to detect the capillary density. After 22 or 15 Gy irradiation, the LV ejection fraction (EF) was impaired in wt mice at 1 week and 4 months after irradiation when compared with sham irradiation. Compared to wt mice, Sestrin2 KO mice had significant reduction in reduced LVEF at 1 week and 4 months after irradiation. A significant increase in LV end-diastolic pressure and myocardial fibrosis and a significant decrease in capillary density were observed in irradiation-wt mice, as well as in irradiation-Sestrin2 KO mice. Sestrin2 involved in the regulation of cardiomyopathy (such as myocardial fibrosis) after irradiation. Overexpression of Sestrin2 might be useful in limiting radiation-induced myocardial injury.

Ligand-based virtual screening and inductive learning for identification of SIRT1 inhibitors in natural products.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase, and its dysregulation can lead to ageing, diabetes, and cancer. From 346 experimentally confirmed SIRT1 inhibitors, an inhibitor structure pattern was generated by inductive logic programming (ILP) with DMax Chemistry Assistant software. The pattern contained amide, amine, and hetero-aromatic five-membered rings, each of which had a hetero-atom and an unsubstituted atom at a distance of 2. According to this pattern, a ligand-based virtual screening of 1 444 880 active compounds from Chinese herbs identified 12 compounds as inhibitors of SIRT1. Three compounds (ZINC08790006, ZINC08792229, and ZINC08792355) had high affinity (-7.3, -7.8, and -8.6 kcal/mol, respectively) for SIRT1 as estimated by molecular docking software AutoDock Vina. This study demonstrated a use of ILP and background knowledge in machine learning to facilitate virtual screening.

Sodium glycididazole enhances the radiosensitivity of laryngeal cancer cells through downregulation of ATM signaling pathway.

The purpose of this study was to evaluate the radiation-enhancing effect of sodium glycididazole, and the corresponding mechanisms of action on laryngeal cancer cells. Two laryngeal cancer cell lines (Hep-2 and UT-SCC-19A) were irradiated with X-rays in the presence or absence of sodium glycididazole. Cell survival, DNA damage and repair, cell apoptosis, cell cycle distribution, expression of proteins related to cell cycle checkpoint, and apoptosis were measured. Significantly increased DNA damages, decreased cells in the G1 phase, arrested cells at G2/M phase, decreased DNA repair protein XRCC1 foci formation, and enhanced cell apoptosis were observed in laryngeal cell lines treated by sodium glycididazole combined with irradiation compared with the irradiation alone. The combined treatment downregulated the protein expressions of ataxia-telangiectasia mutated (ATM), p-ATM, CHK2, and P53 but upregulated the protein expressions of MDM2 and Cdk2. This study indicates that sodium glycididazole enhances the radiosensitivity of laryngeal cancer cells through downregulation of ATM signaling pathway in vitro and in vivo.