CircRNA as an Achilles heel of cancer: characterization, biomarker and therapeutic modalities.

Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs characterized by their lack of 5' caps and 3' poly(A) tails. These molecules have garnered substantial attention from the scientific community. A wide range of circRNA types has been found to be expressed in various tissues of the human body, exhibiting unique characteristics such as high abundance, remarkable stability, and tissue-specific expression patterns. These attributes, along with their detectability in liquid biopsy samples such as plasma, position circRNAs an ideal choice as cancer diagnostic and prognostic biomarkers. Additionally, several studies have reported that the functions of circRNAs are associated with tumor proliferation, metastasis, and drug resistance. They achieve this through various mechanisms, including modulation of parental gene expression, regulation of gene transcription, acting as microRNA (miRNA) sponges, and encoding functional proteins. In recent years, a large number of studies have focused on synthesizing circRNAs in vitro and delivering them to tumor tissue to exert its effects in inhibit tumor progression. Herein, we briefly discuss the biogenesis, characteristics, functions, and detection of circRNAs, emphasizing their clinical potential as biomarkers for cancer diagnosis and prognosis. We also provide an overview the recent techniques for synthesizing circRNAs and delivery strategies, and outline the application of engineered circRNAs in clinical cancer therapy.

A predictive nomogram developed and validated for gastric cancer patients with triple-negative tumor markers.

Aim: To predict the prognosis of gastric cancer patients with triple-negative tumor markers. Materials & methods: Prognostic factors of the nomogram were identified through univariate and multivariate Cox regression analyses. Calibration and receiver operating characteristic curves were used to assess accuracy. Decision curve analysis and concordance indexes were utilized to compare the nomogram with the pathological tumor, node, metastasis stage. Results: A nomogram incorporating log odds of positive lymph nodes, tumor size and lymphocyte-to-monocyte ratio was constructed. The calibration and receiver operating characteristic curves (area under the curve >0.85) showed high accuracy in predicting overall survival. The concordance indexes (0.832 vs 0.760; p < 0.001) and decision curve analysis demonstrated that the nomogram was superior to the pathological tumor, node, metastasis stage. Conclusion: A prediction and risk stratification nomogram has been developed and validated for gastric cancer patients with triple-negative tumor markers.

The Application of Piecewise Regularization Reconstruction to the Calibration of Strain Beams.

Standard beams are mainly used for the calibration of strain sensors using their load reconstruction models. However, as an ill-posed inverse problem, the solution to these models often fails to converge, especially when dealing with dynamic loads of different frequencies. To overcome this problem, a piecewise Tikhonov regularization method (PTR) is proposed to reconstruct dynamic loads. The transfer function matrix is built both using the denoised excitations and the corresponding responses. After singular value decomposition (SVD), the singular values are divided into submatrices of different sizes by utilizing a piecewise function. The regularization parameters are solved by optimizing the piecewise submatrices. The experimental result shows that the MREs of the PTR method are 6.20% at 70 Hz and 5.86% at 80 Hz. The traditional Tikhonov regularization method based on GCV exhibits MREs of 28.44% and 29.61% at frequencies of 70 Hz and 80 Hz, respectively, whereas the L-curve-based approach demonstrates MREs of 29.98% and 18.42% at the same frequencies. Furthermore, the PREs of the PTR method are 3.54% at 70 Hz and 3.73% at 80 Hz. The traditional Tikhonov regularization method based on GCV exhibits PREs of 27.01% and 26.88% at frequencies of 70 Hz and 80 Hz, respectively, whereas the L-curve-based approach demonstrates PREs of 29.50% and 15.56% at the same frequencies. All in all, the method proposed in this paper can be extensively applied to load reconstruction across different frequencies.

[Circular RNA-Encoded Proteins in Gastrointestinal Cancer:A Review].

Circular RNAs(CircRNAs)are a class of non-coding RNAs with a covalently closed-loop structure,high stability,and tissue specificity,with the production mechanisms different from linear RNAs.Recent studies have discovered that some CircRNAs can encode proteins via cap-independent translation mechanisms such as internal ribosome entry site,N6-methyladenosine,and rolling loop translation.The encoded proteins regulate homologous linear proteins or downstream signaling pathways via protein bait or other mechanisms,thereby exerting biological functions.Studies have shown that CircRNAs play a role in various diseases,especially in tumor progression,proliferation,invasion,and metastasis and immune regulation.Therefore,by elucidating the expression and roles of proteins encoded by CircRNAs in tumorigenesis and development,this paper is expected to provide new tumor markers and potential targets for tumor diagnosis and treatment.

Linear Laser Scanning Measurement Method Tracking by a Binocular Vision.

The 3D scanning of a freeform structure relies on the laser probe and the localization system. The localization system, determining the effect of the point cloud reconstruction, will generate positioning errors when the laser probe works in complex paths with a fast speed. To reduce the errors, in this paper, a linear laser scanning measurement method is proposed based on binocular vision calibration. A simple and effective eight-point positioning marker attached to the scanner is proposed to complete the positioning and tracking procedure. Based on this, the method of marked point detection based on image moment and the principle of global coordinate system calibration are introduced in detail. According to the invariance principle of space distance, the corresponding points matching method between different coordinate systems is designed. The experimental results show that the binocular vision system can complete localization under different light intensities and complex environments, and that the repeated translation error of the binocular vision system is less than 0.22 mm, while the rotation error is less than 0.15°. The repeated error of the measurement system is less than 0.36 mm, which can meet the requirements of the 3D shape measurement of the complex workpiece.

Blockchain-based federated learning methodologies in smart environments.

Blockchain technology is an undeniable ledger technology that stores transactions in high-security chains of blocks. Blockchain can solve security and privacy issues in a variety of domains. With the rapid development of smart environments and complicated contracts between users and intelligent devices, federated learning (FL) is a new paradigm to improve accuracy and precision factors of data mining by supporting information privacy and security. Much sensitive information such as patient health records, safety industrial information, and banking personal information in various domains of the Internet of Things (IoT) including smart city, smart healthcare, and smart industry should be collected and gathered to train and test with high potential privacy and secured manner. Using blockchain technology to the adaption of intelligent learning can influence maintaining and sustaining information security and privacy. Finally, blockchain-based FL mechanisms are very hot topics and cut of scientific edge in data science and artificial intelligence. This research proposes a systematic study on the discussion of privacy and security in the field of blockchain-based FL methodologies on the scientific databases to provide an objective road map of the status of this issue. According to the analytical results of this research, blockchain-based FL has been grown significantly during these 5 years and blockchain technology has been used more to solve problems related to patient healthcare records, image retrieval, cancer datasets, industrial equipment, and economical information in the field of IoT applications and smart environments.

Application of a Vision-Based Single Target on Robot Positioning System.

In this paper, we propose a Circular-ring visual location marker based on a global image-matching model to improve the positioning ability in the fiducial marker system of a single-target mobile robot. The unique coding information is designed according to the cross-ratio invariance of the projective theorem. To verify the accuracy of full 6D pose estimation using the Circular-ring marker, a 6 degree of freedom (DoF) robotic arm platform is used to design a visual location experiment. The experimental result shows in terms of small resolution images, different size markers, and long-distance tests that our proposed robot positioning method significantly outperforms AprilTag, ArUco, and Checkerboard. Furthermore, through a repeatable robot positioning experiment, the results indicated that the proposed Circular-ring marker is twice as accurate as the fiducial marker at 2-4 m. In terms of recognition speed, the Circular-ring marker processes a frame within 0.077 s. When the Circular-ring marker is used for robot positioning at 2-4 m, the maximum average translation error of the Circular-ring marker is 2.19, 3.04, and 9.44 mm. The maximum average rotation error is also 1.703°, 1.468°, and 0.782°.

Correction to: Circular RNA circNHSL1 promotes gastric cancer progression through the miR-1306-3p/SIX1/Vimentin axis.

After publication of the article [1], the authors reported errors of inter-duplication in Figure 3.

Circular RNA circCCDC9 acts as a miR-6792-3p sponge to suppress the progression of gastric cancer through regulating CAV1 expression.

BACKGROUND: As a novel type of noncoding RNAs, covalently closed circular RNAs (circRNAs) are ubiquitously expressed in eukaryotes. Emerging studies have related dysregulation of circRNAs to tumorigenesis. However, the biogenesis, regulation, function and mechanism of circRNAs in gastric cancer (GC) remain largely unclear. METHODS: The expression profile of circRNAs in 6 pairs of GC tissues and adjacent non-tumor tissues was analyzed by RNA-sequencing. Quantitative real-time PCR was used to determine the expression level of circCCDC9 in GC tissues and cell lines. Then, functional experiments in vitro and in vivo were employed to explore the effects of circCCDC9 on tumor growth and metastasis in GC. Mechanistically, dual luciferase reporter, fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down assays were performed to confirm that circCCDC9 directly sponged miR-6792-3p and alleviated suppression on target CAV1 expression. RESULTS: Evidently down-regulated expression of circCCDC9 was observed in both GC tissues and cell lines. Expression of circCCDC9 was negatively correlated with tumor size, lymph node invasion, advanced clinical stage and overall survival in GC patients. Functionally, overexpression of circCCDC9 significantly inhibited the proliferation, migration and invasion of GC cell lines in vitro and tumor growth and metastasis in vivo, whereas miR-6792-3p mimics counteracted these effects. Mechanistic analysis demonstrated that circCCDC9 acted as a "ceRNA" of miR-6792-3p to relieve the repressive effect of miR-6792-3p on its target CAV1, then suppressed the tumorigenesis of GC. CONCLUSIONS: CircCCDC9 functions as a tumor suppressor in inhibiting the progression of GC through miR-6792-3p/CAV1 axis, which has provided an exploitable biomarker and therapeutic target for patients with GC.

GINS complex subunit 4, a prognostic biomarker and reversely mediated by Krüppel-like factor 4, promotes the growth of colorectal cancer.

GINS complex subunit 4 (GINS4) is essential for DNA replication initiation and elongation in the G1 /S phase cell cycle in eukaryotes, however, its functional roles and molecular mechanisms remain unclear in many aspects. Our study was designed to investigate the clinical significance, biological function, and molecular mechanism of GINS4 in colorectal cancer (CRC). First, we confirmed that GINS4 expression was significantly overexpressed in CRC cells and tissues. The immunohistochemical results in tissue microarray from 106 CRC patients showed that a high level of GINS4 expression was positively correlated with advanced T stage, higher tumor TNM stage, and poor differentiation. The results from univariate and multivariate survival analysis models based on 106 CRC patients revealed that GINS4 might serve as an independent prognostic indicator for overall survival and disease-free survival of CRC patients. Moreover, downregulated GINS4 can inhibit growth and the cell cycle and accelerate cell apoptosis progression in vitro as well as inhibit tumorigenesis in vivo. Besides, our results also indicated that Krüppel-like factor 4 (KLF4) can negatively regulate GINS4 expression at the transcriptional level and the KLF/GINS4 pathway might play a vital role in the growth and prognosis of CRC.

Circular RNA circNHSL1 promotes gastric cancer progression through the miR-1306-3p/SIX1/vimentin axis.

BACKGROUND: Mounting evidences indicate that circular RNAs (circRNAs) play vital roles in the development and progression of various cancers. However, the detail functions and underlying mechanisms of circRNAs in gastric cancer remain largely unknown. METHODS: The expression profile of metastasis-related circRNAs was screened by RNA-seq analysis. qRT-PCR was used to determine the level and prognostic values of circNHSL1 in gastric cancer tissues. In vitro cell wound healing and transwell (migration and invasion) and in vivo tumorigenesis and metastasis assays were performed to evaluate the functions of circNHSL1. Luciferase reporter, RNA immunoprecipitation (RIP) and rescued assays were employed to confirm the interactions between circNHSL1, miR-1306-3p and SIX1. It's widely accepted that as a mesenchymal marker, Vimentin promotes invasion and metastasis in various cancers. Luciferase reporter assay was used to determine the regulation of SIX1 on Vimentin. In addition, In situ hybridization (ISH) was performed to detect the level and prognostic values of miR-1306-3p. RESULTS: We found that the level of circNHSL1 was significantly up-regulated in gastric cancer, and positively correlated with clinicopathological features and poor prognosis of patients with gastric cancer. Functionally, circNHSL1 promoted cell mobility and invasion, as well as in vivo tumorgenesis and metastasis. Mechanistically, circNHSL1 acted as a miR-1306-3p sponge to relieve the repressive effect of miR-1306-3p on its target SIX1. Moreover, SIX1 enhanced Vimentin expression in the transcriptional level through directly binding to the promoter domain of Vimentin, thereby promoting cell migration and invasion. In addition, miR-1306-3p was down-regulated and negatively correlated with pathological features and poor prognosis in gastric cancer. CONCLUSIONS: CircNHSL1 promotes gastric cancer progression through miR-1306-3p/SIX1/Vimentin axis, and may serve as a novel diagnostic marker and target for treatment of gastric cancer patients.

PODXL, negatively regulated by KLF4, promotes the EMT and metastasis and serves as a novel prognostic indicator of gastric cancer.

BACKGROUND: Podocalyxin-like 1 (PODXL) was reported to be closely associated with the development of various cancers, yet its functional roles and molecular mechanisms remain vague. The aim of our study was to investigate the clinical significance, biological function and molecular mechanism of PODXL in gastric cancer (GC). METHODS: The level of PODXL in GC tissues was detected applying GC tissues microarray, fresh GC tissues and public database (Oncomine). The invasion, metastasis and tumorigenesis role of PODXL were performed in vitro and in vivo. The correlations between KLF4 and PODXL was determined in GC tissues microarray and fresh GC tissues, and the molecular regulatory mechanism between KLF4 and PODXL was explored in vitro. RESULTS: The high level of PODXL was detected in GC tissues with advanced T stage, lymph node metastasis, Union for International Cancer Control stage and poor differentiation. And Cox proportional hazards model revealed that PODXL can serve as an independent prognostic indicator for disease-free survival and overall survival of GC patients. Moreover, downregulation of PODXL could inhibit EMT and reduce invasion and metastasis in vitro as well as tumorigenesis in vivo. Additionally, our findings showed that PODXL may be a significant downstream target of KLF4. CONCLUSIONS: KLF4/PODXL signaling pathway assumes an irreplaceable role in tumorigenesis, invasion and metastasis of human GC and PODXL serves as an independent prognostic indicator for GC patients.

Iodine-catalyzed direct C-H thiolation of imidazo[1,5-a]quinolines for the synthesis of 3-sulfenylimidazo[1,5-a]quinolines.

An iodine-catalyzed regioselective sulfenylation of imidazo[1,5-a]quinolines was developed under metal- and oxidant-free reaction conditions. Using disulfides or thiophenols as sulfenylating agents, 3-sulfenylimidazo[1,5-a]quinoline derivatives were obtained in good to excellent yields with broad functional group tolerance. A multi-component reaction to generate 1-sulfenylated imidazo[1,5-a]pyridines is also described. Preliminary biological evaluation showed that some of the 3-sulfenylated imidazo[1,5-a]quinolines had significant anticancer activity.

Copper-Promoted Double Oxidative C-H Amination Cascade for the Synthesis of Imidazo[1,5-a]quinolines.

A copper-promoted cascade reaction of 2-methylazaarenes and benzylamines has been developed via sequential double oxidative C(sp(3))-H aminations. This protocol provides straightforward access to imidazo[1,5-a]quinoline derivatives without employing prefunctionalized substrates.

Insights into the role of glycosyltransferase in the targeted treatment of gastric cancer.

Gastric cancer is a remarkably heterogeneous tumor. Despite some advances in the diagnosis and treatment of gastric cancer in recent years, the precise treatment and curative outcomes remain unsatisfactory. Poor prognosis continues to pose a major challenge in gastric cancer. Therefore, it is imperative to identify effective targets to improve the treatment and prognosis of gastric cancer patients. It should be noted that glycosylation, a novel form of posttranslational modification, is a process capable of regulating protein function and influencing cellular activities. Currently, numerous studies have shown that glycosylation plays vital roles in the occurrence and progression of gastric cancer. As crucial enzymes that regulate glycan synthesis in glycosylation processes, glycosyltransferases are potential targets for treating GC. Hence, investigating the regulation of glycosyltransferases and the expression of associated proteins in gastric cancer cells is highly important. In this review, the related glycosyltransferases and their related signaling pathways in gastric cancer, as well as the existing inhibitors of glycosyltransferases, provide more possibilities for targeted therapies for gastric cancer.

Insights into the history and tendency of glycosylation and digestive system tumor: A bibliometric-based visual analysis.

BACKGROUND: Glycosylation, a commonly occurring post-translational modification, is highly expressed in several tumors, specifically in those of the digestive system, and plays a role in various cellular pathophysiological mechanisms. Although the importance and detection methods of glycosylation in digestive system tumors have garnered increasing attention in recent years, bibliometric analysis of this field remains scarce. The present study aims to identify the developmental trends and research hotspots of glycosylation in digestive system tumors. AIM: To find and identify the developmental trends and research hotspots of glycosylation in digestive system tumors. METHODS: We obtained relevant literature from the Web of Science Core Collection and employed VOSviewer 1.6.19 and CiteSpace (version 6.1.R6) to perform bibliometric analysis. RESULTS: A total of 2042 documents spanning from 1978 to the present were analyzed, with the research process divided into three phases: the period of obscurity (1978-1990), continuous development period (1991-2006), and the rapid outbreak period (2007-2023). These documents were authored by researchers from 66 countries or regions, with the United States and China leading in terms of publication output. Reis Celso A had the highest number of publications, while Pinho SS was the most cited author. Co-occurrence analysis revealed the most popular keywords in this field are glycosylation, expression, cancer, colorectal cancer, and pancreatic cancer. Furthermore, the Journal of Proteome Research was the most prolific journal in terms of publications, while the Journal of Biological Chemistry had the most citations. CONCLUSION: The bibliometric analysis shows current research focus is primarily on basic research in this field. However, future research should aim to utilize glycosylation as a target for treating tumor patients.

The transition of surgical simulation training and its learning curve: a bibliometric analysis from 2000 to 2023.

BACKGROUND: Proficient surgical skills are essential for surgeons, making surgical training an important part of surgical education. The development of technology promotes the diversification of surgical training types. This study analyzes the changes in surgical training patterns from the perspective of bibliometrics, and applies the learning curves as a measure to demonstrate their teaching ability. METHOD: Related papers were searched in the Web of Science database using the following formula: TS=[(training OR simulation) AND (learning curve) AND (surgical)]. Two researchers browsed the papers to ensure that the topics of articles were focused on the impact of surgical simulation training on the learning curve. CiteSpace, VOSviewer, and R packages were applied to analyze the publication trends, countries, authors, keywords, and references of selected articles. RESULT: Ultimately, 2461 documents were screened and analyzed. The USA is the most productive and influential country in this field. Surgical endoscopy and other interventional techniques publish the most articles, while surgical endoscopy and other interventional techniques is the most cited journal. Aggarwal Rajesh is the most productive and influential author. Keyword and reference analyses reveal that laparoscopic surgery, robotic surgery, virtue reality, and artificial intelligence were the hotspots in the field. CONCLUSION: This study provided a global overview of the current state and future trend in the surgical education field. The study surmised the applicability of different surgical simulation types by comparing and analyzing the learning curves, which is helpful for the development of this field.

A predictive model and rapid multi-dynamic algorithm developed based on tumor-stroma percentage in gastric cancer: a retrospective, observational study.

Background: Tumor-stroma percentage (TSP) is a prognostic risk factor in numerous solid tumors. Despite this, the prognostic significance of TSP in gastric cancer (GC) remains underexplored. Through the development of a personalized predictive model and a semi-automatic identification system, our study aimed to fully unlock the predictive potential of TSP in GC. Methods: We screened GC patients from Shanghai General Hospital (SGH) between 2012 and 2019 to develop and validate a nomogram. Univariate and multivariate Cox proportional hazards regression analyses were employed to identify independent prognostic factors influencing the prognosis for GC patients. The nomogram was further validated externally by using a cohort from Bengbu Medical College (BMC). All patients underwent radical gastrectomy, with those diagnosed with locally advanced GC receiving adjuvant chemotherapy. The primary outcome measured was overall survival (OS). The semi-automatic identification of the TSP was achieved through a computer-aided detection (CAD) system, denoted as TSP-cad, while TSP identified by pathologists was labeled as TSP-visual. Results: A total of 813 GC patients from SGH and 59 from BMC were enrolled in our study. TSP-visual was identified as an adverse prognostic factor for OS in GC and was found to be associated with pathological Tumor Node Metastasis staging system (pTNM) stage, T stage, N stage, perineural invasion (PNI), lymphovascular invasion (LVI), TSP-visual, tumor size, and other factors. Multivariate Cox regression using the training cohort revealed that TSP-visual (hazard ratio [HR], 2.042; 95% confidential interval [CI], 1.485-2.806; P < 0.001), N stage (HR, 2.136; 95% CI, 1.343-3.397; P = 0.010), PNI (HR , 1.791; 95% CI, 1.270-2.526; P = 0.001), and LVI (HR, 1.482; 95% CI, 1.021-2.152; P = 0.039) were independent predictors. These factors were incorporated into a novel nomogram, which exhibited strong predictive accuracy for 5-year OS in the training, internal validation, and external validation cohorts (area under the curve = 0.744, 0.759, and 0.854, respectively). The decision curve analysis of the nomogram and concordance indexes across the three cohorts outperformed the traditional pTNM (P < 0.05). Additionally, TSP-cad assessment using a rapid multi-dynamic algorithm demonstrated good agreement with TSP-visual. Conclusions: The novel nomogram based on TSP could effectively identify individuals at risk of a poor prognosis among patients with GC. TSP-cad is anticipated to enhance the evaluation process of TSP.

Waterlogging in soil restricts the growth of Gleditsia sinensis seedlings and inhibits the accumulation of lignans and phenolic acids in thorns.

Gleditsia sinensis, commonly known as Chinese Zaojiao, has important economic value and medicinal compounds in its fruits and thorns, making it widely cultivated artificially in China. However, the available literature on the impact of waterlogging on the growth of G. sinensis seedlings and the accumulation of metabolite compounds in its thorns is limited. To address this knowledge gap, G. sinensis seedlings were planted in soil supplemented with pindstrup substrate, which enhances the water-holding capacity of the soil. The analyses of morphological traits and nutrient elements in one-year-old G. sinensis seedlings grown naturally under ambient conditions and metabolite accumulation in its thorns were conducted. The results showed that the waterlogged soil significantly diminished the height, fresh weight, and dry weight of seedling roots and stems (P < 0.05). Furthermore, waterlogging hindered the uptake of iron (Fe) and manganese (Mn), as well as the transport of potassium (K). The identified metabolites within the thorns were categorized into 16 distinct groups. Relative to the control soil, fatty acids and derivatives were the most down-regulated metabolites in the waterlogged soil, accounting for 40.58% of the total metabolites, followed by lignans (38.71%), phenolic acids (34.48%), saccharides and alcohols (34.15%), steroids (16.67%), alkaloids (12.24%), flavonoids (9.28%), and glycerophospholipids (7.41%). Conversely, nucleotides and derivatives experienced the greatest up-regulation in the waterlogged soil, accounting for 50.00% of the total metabolites. In conclusion, waterlogging negatively impacted the growth of G. sinensis seedlings and inhibited the accumulation of metabolites. Hence, when considering the accumulation of secondary metabolites such as lignans and phenolic acids, appropriate management of soil moisture levels should be taken into account.

Novel hypoxia-induced HIF1α-circTDRD3-positive feedback loop promotes the growth and metastasis of colorectal cancer.

Tumor hypoxia and circular RNAs (circRNAs) are considered to play key roles in tumor progression and malignancy, respectively. Nevertheless, the biological functions and underlying mechanisms of specific circRNAs exposed to hypoxic microenvironments in colorectal cancer (CRC) remain largely elusive. Herein, a novel circRNA, circTDRD3, which is upregulated under hypoxic conditions, was identified. The expression of circTDRD3 was highly expressed in CRC tissues and positively correlated with overall survival, tumor size, lymph node invasion and clinical stage. CircTDRD3 facilitated CRC cell proliferation, migration and metastasis in vitro and in vivo. Mechanistically, circTDRD3 promoted HIF1α expression by sponging miR-1231, which facilitated CRC progression. Meanwhile, HIF1α directly combined with TDRD3 promoter to increase the expression of TDRD3 pre-mRNA. Then HIF1a-induced PTBP1 accelerated the formation of circTDRD3. Our findings reveal that circTDRD3 facilitates the proliferation and metastasis of CRC through a positive feedback loop mediated by the HIF1α/PTBP1/circTDRD3/miR-1231/HIF1α axis. Therefore, circTDRD3 may serve as a prognostic biomarker and therapeutic target for patients with CRC.