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Objective: Recently, the application in the field of germplasm resource conservation has become an important application of primordial germ cells (PGCs). However, due to the lack of deep understanding of the biological characteristics of PGCs at different time points, there is no systematic scheme for the selection of PGCs at which time points in practical application, which affects the practical application effect of PGCs. This study aims to clarify the differences in PGCs during development. Methods: Here, migration experiment, EdU proliferation assay and cell apoptosis assay were conducted to compare the differences in the migration ability, the proliferation ability and the recovery efficiency among female and male PGCs at E3.5, E4.5 and E5.5, which were explained by the following transcriptome sequencing analysis. Results: We found that there were larger differences between female and male PGCs at different embryonic ages, while smaller differences between female and male PGCs at the same embryonic age. Further comparison showed that the cell migration ability of female and male PGCs decreased gradually during development, so female and male PGCs at E3.5 are more suitable for in vitro allotransplantation. At the same time, the proliferation ability of PGCs gradually decreased during development, and cell adhesion and extracellular matrix communication were weakened, indicating that female and male PGCs of E3.5 are more suitable for in vitro long-term culture cell line establishment. Interestingly, female and male PGCs at E5.5 showed strong DNA damage repair ability, thus more suitable for in vitro long-term cryopreservation. Conclusion: This study provides a theoretical basis for systematically selecting PGCs at suitable developmental time points as cell materials for efficient utilization by analyzing the characteristics of female and male PGCs at different developmental time points based on transcriptome.
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In chicken, primordial germ cells (PGC) are crucial for the preservation and manipulation of genetic resources in poultry production. The HiS and FAcs culture systems are two important methods for the in vitro cultivation of chicken PGCs. The purpose of this study was to compare and analyze the two cultivation systems for PGCs (His and FAcs culture systems) to assess their efficacy and applicability in supporting PGC growth, maintaining PGC characteristics, and lineage transmission ability. The study found that both HiS and FAcs culture systems could maintain the basic biological characteristics of chicken PGCs, including the simultaneous expression of pluripotency and reproductive marker genes, as well as the presence of abundant glycogen granules. Subsequently, we identified 2,145 differentially expressed genes (DEG) through RNA sequencing. GO and KEGG analysis revealed a large number of DEGs enriched in the cell adhesion and calcium ion binding pathways, and the analysis found that these genes maintained a higher level in HiS-PGCs. Further personalized analysis found that the regulatory genes for maintaining PGC pluripotency were highly expressed in HiS-PGCs, while germ cell-related genes showed similar expression in both systems. Additionally, through RNA sequencing data and cell proliferation ability, it was found that PGCs in the FAcs system had a higher proliferation rate and a faster cell cycle. Finally, it was discovered that the expression of cell migration-related genes was maintained at a higher level in HiS-PGCs, but the migration efficiency of HiS-PGCs did not show a significant difference compared to FAcs-PGCs. These results suggest that both HiS and FAcs culture systems can maintain the proliferation and basic characteristics of chicken PGCs, but differences exist in cell proliferation, pluripotency regulation, and cell adhesion. These findings provide new information for optimizing PGC cultivation systems and are important for the preservation and genetic improvement of chicken PGCs.
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Carrier-phase noise limits both the performance and the maximum operation range of coherent LiDAR. To address this issue, we propose a carrier-phase-noise-canceled LiDAR based on an auxiliary interferometer and adaptive filters. Compared to previous methods, this approach is calibration-free and offers higher compensation accuracy, as well as applicability of dynamic target detection. Experiments of range-Doppler imaging for stationary targets and rotating extended targets have been performed, and the detection results close to the theoretical resolution were obtained at the round trip distance to the target beyond 981 times and 106 times coherence length, respectively.
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The incorporation of automatic segmentation methodologies into dental X-ray images refined the paradigms of clinical diagnostics and therapeutic planning by facilitating meticulous, pixel-level articulation of both dental structures and proximate tissues. This underpins the pillars of early pathological detection and meticulous disease progression monitoring. Nonetheless, conventional segmentation frameworks often encounter significant setbacks attributable to the intrinsic limitations of X-ray imaging, including compromised image fidelity, obscured delineation of structural boundaries, and the intricate anatomical structures of dental constituents such as pulp, enamel, and dentin. To surmount these impediments, we propose the Deformable Convolution and Mamba Integration Network, an innovative 2D dental X-ray image segmentation architecture, which amalgamates a Coalescent Structural Deformable Encoder, a Cognitively-Optimized Semantic Enhance Module, and a Hierarchical Convergence Decoder. Collectively, these components bolster the management of multi-scale global features, fortify the stability of feature representation, and refine the amalgamation of feature vectors. A comparative assessment against 14 baselines underscores its efficacy, registering a 0.95% enhancement in the Dice Coefficient and a diminution of the 95th percentile Hausdorff Distance to 7.494.
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Processamento de Imagem Assistida por Computador , Humanos , Processamento de Imagem Assistida por Computador/métodos , Redes Neurais de Computação , Algoritmos , Dente/diagnóstico por imagemRESUMO
Purpose: Fundus fluorescein angiography (FFA) is the gold standard for retinal vein occlusion (RVO) diagnosis. This study aims to develop a deep learning-based system to diagnose and classify RVO using FFA images, addressing the challenges of time-consuming and variable interpretations by ophthalmologists. Methods: 4028 FFA images of 467 eyes from 463 patients were collected and annotated. Three convolutional neural networks (CNN) models (ResNet50, VGG19, InceptionV3) were trained to generate the label of image quality, eye, location, phase, lesions, diagnosis, and macular involvement. The performance of the models was evaluated by accuracy, precision, recall, F-1 score, the area under the curve, confusion matrix, human-machine comparison, and Clinical validation on three external data sets. Results: The InceptionV3 model outperformed ResNet50 and VGG19 in labeling and interpreting FFA images for RVO diagnosis, achieving 77.63%-96.45% accuracy for basic information labels and 81.72%-96.45% for RVO-relevant labels. The comparison between the best CNN and ophthalmologists showed up to 19% accuracy improvement with the inceptionV3. Conclusion: This study developed a deep learning model capable of automatically multi-label and multi-classification of FFA images for RVO diagnosis. The proposed system is anticipated to serve as a new tool for diagnosing RVO in places short of medical resources.
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The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation.
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Galinhas , Edição de Genes , Cromossomos Sexuais , Animais , Galinhas/genética , Edição de Genes/métodos , Cromossomos Sexuais/genética , Mutagênese Insercional , Sistemas CRISPR-Cas , Linhagem Celular , Técnicas de Inativação de Genes/métodos , Feminino , MasculinoRESUMO
BACKGROUND: Short-term (5 min) heart rate variability (HRV) analysis is widely used in assessing autonomic nervous system activity during exercise. While shortening the HRV measurement duration can help improve its application efficiency, its accuracy needs to be verified. This study investigated the agreement between ultra-short-term (UST) HRV (3 or 4 min) and standard 5 min HRV and explored the optimal recording duration under resting and post-exercise conditions. METHODS: Fourteen participants exercised on a cycle ergometer at 60% of their maximum peak power. Data were collected during the rest condition (Pre-E) and three post-exercise conditions (Post-E1, Post-E2, and Post-E3), with indicators of the standard deviation (SDNN) of the ultra-short and short-term RR intervals and the root mean square (RMSSD) of the continuous difference between RR intervals. Repeated measures ANOVA, Cohen's d statistic, Bland-Altman analysis, and interclass correlation coefficients (ICC) assessed the agreement between UST-HRV and ST-HRV. RESULTS: The consistency results of SDNN and RMSSD in resting and post-exercise were different. At the Pre-E, Post-E2, and Post-E3 phases, no statistical differences for SDNN and RMSSD were observed, with ICCs surpassing 0.9, indicating a high level of agreement. However, at Post-E2, there was a significant difference between 3 min RMSSD and 5 min RMSSD (p < 0.05), as well as between 3 min SDNN, 4 min SDNN, and 5 min SDNN (p < 0.05). Furthermore, the limits of agreement were observed to decrease as the time duration increased in Bland-Altman plots. CONCLUSIONS: UST-HRV analysis is a reliable substitute for standard 5 min HRV assessment, particularly during resting conditions. For post-exercise measurements, assessing the appropriateness of a 3- or 4 min duration based on the exercise's length is recommended to ensure accuracy and reliability.
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As an RNA binding protein (RBP), DDX5 is widely involved in the regulation of various biological activities. While recent studies have confirmed that DDX5 can act as a transcriptional cofactor that is involved in the formation of gametes, few studies have investigated whether DDX5 can be used as a transcription factor to regulate the formation of primordial germ cells (PGCs). In this study, we found that DDX5 was significantly up-regulated during chicken PGC formation. Under different PGC induction models, the overexpression of DDX5 not only up-regulates PGC markers but also significantly improves the formation efficiency of primordial germ cell-like cells (PGCLC). Conversely, the inhibition of DDX5 expression can significantly inhibit both the expression of PGC markers and PGCLC formation efficiency. The effect of DDX5 on PGC formation in vivo was consistent with that seen in vitro. Interestingly, DDX5 not only participates in the formation of PGCs but also positively regulates their migration and proliferation. In the process of studying the mechanism by which DDX5 regulates PGC formation, we found that DDX5 acts as a transcription factor to bind to the promoter region of BMP4-a key gene for PGC formation-and activates the expression of BMP4. In summary, we confirm that DDX5 can act as a positive transcription factor to regulate the formation of PGCs in chickens. The obtained results not only enhance our understanding of the way in which DDX5 regulates the development of germ cells but also provide a new target for systematically optimizing the culture and induction system of PGCs in chickens in vitro.
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Proteína Morfogenética Óssea 4 , Galinhas , RNA Helicases DEAD-box , Células Germinativas , Animais , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/genética , Células Germinativas/metabolismo , Galinhas/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proliferação de Células , Movimento Celular/genética , Regiões Promotoras GenéticasRESUMO
To address the increasing issue of antibiotic wastewater, this study applied a static magnetic field (SMF) to the activated sludge process to increase the efficiency of tetracycline (TC) removal from swine wastewater and to reveal its enhanced mechanisms. The results demonstrated that the SMF-modified activated sludge process could achieve almost complete TC removal at sludge loading rates of 0.3 mg TC/g MLSS/d. Analysis of zeta potential and extracellular polymeric substances composition of the activated sludge revealed that SMF increased electrostatic interactions between TC and activated sludge and made activated sludge has much more binding sites, finally resulting in the increased TC biosorption. Metagenomic analysis showed that SMF promoted the enrichment of ammonia-oxidizing bacteria, TC-degrading bacteria, and aromatic compounds-degrading bacteria; it also enhanced ammonia monooxygenase- and cytochrome P450-mediated TC metabolism while upregulating functional genes associated with oxidase, reductase, and dehydrogenase - all contributing to increased TC biodegradation. Additionally, SMF mitigated the enrichment and spread of antibiotic resistance genes (ARGs) by decreasing the abundance of potential hosts of ARGs and inhibiting the upregulation of genes encoding ABC transporters and putative transposase. Based on these findings, this study demonstrates that magnetic field is an enhancement strategy with great potential to relieve the harmful impacts of the growing antibiotic wastewater problem on human health and the ecosystem.
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Resistência Microbiana a Medicamentos , Campos Magnéticos , Esgotos , Tetraciclina , Tetraciclina/farmacologia , Resistência Microbiana a Medicamentos/genética , Antibacterianos/farmacologia , Águas Residuárias/química , Animais , Eliminação de Resíduos Líquidos/métodos , Bactérias/genética , Bactérias/metabolismo , Biodegradação Ambiental , SuínosRESUMO
Background: Lung adenocarcinoma (LUAD) is among the most prevalent malignancies worldwide, with unfavorable treatment outcomes. Peptidyl-prolyl isomerase F (PPIF) is known to influence the malignancy traits of tumor progression by modulating the bioenergetics and mitochondrial permeability in cancer cells; however, its role in LUAD remains unclear. Our study seeks to investigate the clinical significance, tumor proliferation, and immune regulatory functions of PPIF in LUAD. Methods: The expression of PPIF in LUAD tissues and cells was assessed using bioinformatics analysis, immunohistochemistry (IHC), and Western blotting. Survival curve analysis was conducted to examine the prognostic association between PPIF expression and LUAD. The immunomodulatory role of PPIF in LUAD was assessed through the analysis of PPIF expression and immune cell infiltration. A series of gain- and loss-of-function experiments were conducted on PPIF to investigate its biological functions in LUAD both in vitro and in vivo. The mechanisms underlying PPIF's effects on LUAD were delineated through functional enrichment analysis and Western blotting assays. Results: PPIF exhibited overexpression in LUAD tissues compared to normal controls. Survival curve analysis revealed that patients with LUAD exhibiting higher PPIF expression demonstrated decreased overall survival and a shorter progression-free interval. PPIF was implicated in modulating immune cell infiltration, particularly in regulating the T helper 1-T helper 2 cell balance. Functionally, PPIF was discovered to promote tumor cell proliferation and advance cell-cycle progression. Furthermore, PPIF could impede mitophagy by targeting the FOXO3a/PINK1-Parkin signaling pathway. Conclusions: The findings of this study indicate that the prognosis-related gene PPIF may have a significant role in the regulation of LUAD cell proliferation, tumor-associated immune cell infiltration, and mitophagy, and thus PPIF may be a promising therapeutic target of LUAD.
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Background: Lung adenocarcinoma (LUAD) is one of the most common types of cancer worldwide. Proteasome activator subunit 3 (PSME3) is a subunit of a proteasome activator, and changes in PSME3 can lead to the development of many diseases in organisms. However, the specific mechanism of PSME3 in LUAD has not yet been elucidated. This study initially revealed the mechanism of PSME3 promoting the progression of lung adenocarcinoma, which provided a potential molecular target for clinical treatment. Methods: PSME3 expression in LUAD cells and tissues was assessed by bioinformatics analysis, immunohistochemistry (IHC), Western blotting (WB), and quantitative real time polymerase chain reaction (qRT-PCR). A series of functional experiments were used to evaluate the effects of PSME3 knockdown and overexpression on LUAD cell proliferation, migration, and apoptosis. The potential mechanism of PSME3 was explored by transcriptome sequencing and WB experiments. Results: In this study, our initial findings indicated that PSME3 expression was abnormally high in LUAD and was associated with poor patient prognosis. Further, we found that the downregulation of PSME3 significantly inhibited LUAD cell proliferation, an effect that was verified by subcutaneous tumor formation experiments in nude mice. Similarly, the rate of invasion and migration of LUAD cells significantly decreased after the downregulation of PSME3. Using flow cytometry, we found that the knockdown of PSME3 caused cell cycle arrest at the G1/S phase. Through transcriptome sequencing, we found that the transforming growth factor-beta (TGF-ß)/SMAD signaling pathway was closely related to LUAD, and we then validated the pathway using WB assays. Conclusions: We demonstrated that PSME3 was abnormally highly expressed in LUAD and related to poor patient prognosis; therefore, targeting PSME3 in the treatment of LUAD may represent a novel therapeutic approach.
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Conventional adhesives experience reduced adhesion when exposed to aqueous environments. The development of underwater adhesives capable of forming strong and durable bonds across various wet substrates is crucial in biomedical and engineering domains. Nonetheless, limited emphasis placed on retaining high adhesion strengths in different saline environments, addressing challenges such as elevated osmotic pressure and spontaneous dimensional alterations. Herein, a series of ionogel-based underwater adhesives are developed using a copolymerization approach that incorporates "dynamic complementary cross-linking" networks. Synergistic engineering of building blocks, cross-linking networks, pendant groups and counterions within ionogels ensures their adhesion and cohesion in brine spanning a wide salinity range. A high adhesion strength of ≈3.6 MPa is attained in freshwater. Gratifyingly, steady adhesion strengths exceeding 3.3 MPa are retained in hypersaline solutions with salinity ranging from 50 to 200 g kg-1, delivering one of the best-performing underwater adhesives suitable for diverse saline solutions. A combination of outstanding durability, reliability, deformation resistance, salt tolerance, and self-healing properties showcases the "self-contained" underwater adhesion. This study shines light on the facile fabrication of catechol-free ionogel-based adhesives, not merely boosting adhesion strengths in freshwater, but also broadening their applicability across various saline environments.
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7-Aminoindoles are important synthetic intermediates to a broad range of bioactive molecules. Transition metal-catalyzed directed C-H amination is among the most straightforward route for their synthesis, whereas methods that could directly incorporate an NH2 group in a highly selective manner remains elusive. Moreover, there is still high demand for the development of earth-abundant metal catalysis for such attractive reactivity. We present here the first C-7 selective NH2 amination of indoles through a directed homolytic aromatic substitution (HAS) with iron-aminyl radical. The reaction exhibits broad substrate scope, tolerates variety of functional groups, and is readily scalable with catalyst loading down to 0.1 mol% and turnover number (TON) up to 4500.
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Retinal neovascularization (RNV), a typical pathological manifestation involved in most neovascular diseases, causes retinal detachment, vision loss, and ultimately irreversible blindness. Repeated intravitreal injections of anti-VEGF drugs were developed against RNV, with limitations of incomplete responses and adverse effects. Therefore, a new treatment with a better curative effect and more prolonged dosage is demanding. Here, we induced macrophage polarization to anti-inflammatory M2 phenotype by inhibiting cGAS-STING signaling with an antagonist C176, appreciating the role of cGAS-STING signaling in the retina in pro-inflammatory M1 polarization. C176-loaded and phosphatidylserine-modified dendritic mesoporous silica nanoparticles were constructed and examined by a single intravitreal injection. The biosafe nanoparticles were phagocytosed by retinal macrophages through a phosphatidylserine-mediated "eat me" signal, which persistently release C176 to suppress STING signaling and thereby promote macrophage M2 polarization specifically. A single dosage can effectively alleviate pathological angiogenesis phenotypes in murine oxygen-induced retinopathy models. In conclusion, these C176-loaded nanoparticles with enhanced cell uptake and long-lasting STING inhibition effects might serve as a promising way for treating RNV.
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Background: The convergence of smartphone technology and artificial intelligence (AI) has revolutionized the landscape of ophthalmic care, offering unprecedented opportunities for diagnosis, monitoring, and management of ocular conditions. Nevertheless, there is a lack of systematic studies on discussing the integration of smartphone and AI in this field. Main text: This review includes 52 studies, and explores the integration of smartphones and AI in ophthalmology, delineating its collective impact on screening methodologies, disease detection, telemedicine initiatives, and patient management. The collective findings from the curated studies indicate promising performance of the smartphone-based AI screening for various ocular diseases which encompass major retinal diseases, glaucoma, cataract, visual impairment in children and ocular surface diseases. Moreover, the utilization of smartphone-based imaging modalities, coupled with AI algorithms, is able to provide timely, efficient and cost-effective screening for ocular pathologies. This modality can also facilitate patient self-monitoring, remote patient monitoring and enhancing accessibility to eye care services, particularly in underserved regions. Challenges involving data privacy, algorithm validation, regulatory frameworks and issues of trust are still need to be addressed. Furthermore, evaluation on real-world implementation is imperative as well, and real-world prospective studies are currently lacking. Conclusions: Smartphone ocular imaging merged with AI enables earlier, precise diagnoses, personalized treatments, and enhanced service accessibility in eye care. Collaboration is crucial to navigate ethical and data security challenges while responsibly leveraging these innovations, promising a potential revolution in care access and global eye health equity.
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We conducted a systematic review of research in artificial intelligence (AI) for retinal fundus photographic images. We highlighted the use of various AI algorithms, including deep learning (DL) models, for application in ophthalmic and non-ophthalmic (i.e., systemic) disorders. We found that the use of AI algorithms for the interpretation of retinal images, compared to clinical data and physician experts, represents an innovative solution with demonstrated superior accuracy in identifying many ophthalmic (e.g., diabetic retinopathy (DR), age-related macular degeneration (AMD), optic nerve disorders), and non-ophthalmic disorders (e.g., dementia, cardiovascular disease). There has been a significant amount of clinical and imaging data for this research, leading to the potential incorporation of AI and DL for automated analysis. AI has the potential to transform healthcare by improving accuracy, speed, and workflow, lowering cost, increasing access, reducing mistakes, and transforming healthcare worker education and training.
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Embryonic stem cells (ESCs) are remarkably undifferentiated cells that originate from the inner cell mass of the blastocyst. They possess the ability to self-renew and differentiate into multiple cell types, making them invaluable in diverse applications such as disease modeling and the creation of transgenic animals. In recent years, as agricultural practices have evolved from traditional to biological breeding, it has become clear that pluripotent stem cells (PSCs), either ESCs or induced pluripotent stem cells (iPSCs), are optimal for continually screening suitable cellular materials. However, the technologies for long-term in vitro culture or establishment of cell lines for PSCs in livestock are still immature, and research progress is uneven, which poses challenges for the application of PSCs in various fields. The establishment of a robust in vitro system for these cells is critically dependent on understanding their pluripotency maintenance mechanisms. It is believed that the combined effects of pluripotent transcription factors, pivotal signaling pathways, and epigenetic regulation contribute to maintaining their pluripotent state, forming a comprehensive regulatory network. This article will delve into the primary mechanisms underlying the maintenance of pluripotency in PSCs and elaborate on the applications of PSCs in the field of livestock.
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Mechanotransduction is the essential process that cells convert mechanical force into biochemical responses, and electrochemical sensor stands out from existing techniques by providing quantitative and real-time information about the biochemical signals during cellular mechanotransduction. However, the intracellular biochemical response evoked by mechanical force has been poorly monitored. In this paper, we report a method to apply local stretch on single cell and simultaneously monitor the ensuing intracellular biochemical signals. Specifically, a ferromagnetic micropipette was fabricated to locally stretch a single cell labeled with Fe3O4 nanoparticles under the external magnetic field, and the SiC@Pt nanowire electrode (SiC@Pt NWE) was inserted into the cell to monitor the intracellular hydrogen peroxide (H2O2) production induced by the local stretch. As a proof of concept, this work quantitatively investigated the elevated amount of H2O2 levels in single endothelial cell under different stretching amplitudes. This work puts forward a new research modality to manipulate and monitor the mechanotransduction at the single-cell level.
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Peróxido de Hidrogênio , Mecanotransdução Celular , Nanofios , Análise de Célula Única , Peróxido de Hidrogênio/análise , Análise de Célula Única/métodos , Mecanotransdução Celular/fisiologia , Nanofios/química , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Humanos , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Platina/química , EletrodosRESUMO
Exocytosis involving the fusion of intracellular vesicles with cell membrane, is thought to be modulated by the mechanical cues in the microenvironment. Single-cell electrochemistry can offer unique information about the quantification and kinetics of exocytotic events; however, the effects of mechanical force on vesicular release have been poorly explored. Herein, we developed a stretchable microelectrode with excellent electrochemical stability under mechanical deformation by microfabrication of functionalized poly(3,4-ethylenedioxythiophene) conductive ink, which achieved real-time quantitation of strain-induced vesicular exocytosis from a single cell for the first time. We found that mechanical strain could cause calcium influx via the activation of Piezo1 channels in chromaffin cell, initiating the vesicular exocytosis process. Interestingly, mechanical strain increases the amount of catecholamines released by accelerating the opening and prolonging the closing of fusion pore during exocytosis. This work is expected to provide revealing insights into the regulatory effects of mechanical stimuli on vesicular exocytosis.