Segment Anything Models (SAMs) generalize well for generic vision tasks; however, they underperform on ultrasound images. Moreover, they require extensive annotated data for training. To address their limitations in generalization, high training data demands, and computational resource usage on ultrasound segmentation, we propose UltraSAM, a foundational medical ultrasound segmentation model. The main contributions of the study are as follows: First, we propose a Fast Fourier Transform (FFT) Convolutional (FFTConv) branch to enhance local frequency-domain feature extraction from ultrasound images, enabling better learning of generalizable ultrasound features. Second, we introduce an FFT injector (FFTI) to promote feature interaction between spatial and frequency domains, reducing training data requirements and improving the generalization of SAM on ultrasound images. Finally, low-rank adapters (LoRAs) are used to fine-tune the encoder, preserving the general visual representation of SAM while enabling domain adaptation for ultrasound imaging with minimal computational cost. We demonstrated the effectiveness of UltraSAM across four downstream segmentation tasks on 10 publicly available datasets spanning eight different ultrasound modalities. UltraSAM outperformed nine task-specific methods and six SAM-based foundation methods with only hundreds of annotated training samples. Compared with existing SAM-based segmentation methods, UltraSAM achieved a 2.07%-59.45% improvement in the Dice score in zero-shot segmentation tasks. In summary, UltraSAM enhances ultrasound diagnostics and personalized therapeutics by reducing dependence on resource-intensive datasets, holding potential to be a foundational clinical decision-support system.

Background Multi-modal ultrasonic images method for grading prostate cancer is becoming an effective diagnostic technique. However, the subjective integration of images’ information leads to grade variability between pathologists, potentially impacting clinical decision-making. Herein, we developed a multimodal adaptive feature fusion network (MAFF-Net) as a computer-aided network system for prostate cancer classification and grading. Methods We proposed and designed two core modules in the MAFF-Net, which include the triple efficient feature extractor (TEFE) and the adaptive feature fusion module (AFFM). The proposed TEFE extracts multiple features from B-mode Ultrasound (BUS), Color Doppler Flow Imaging (CDFI), and Ultrasound Elastography (UE) images, which include tissue microstructure, hemodynamic changes, and mechanical elasticity characteristics. The AFFM adaptively assigns weights to features from different modalities via an attention mechanism. A multi-modal ultrasonic images prostate database was established for training and validating, which included 870 cross-sectional images collected from 182 patients. Results The proposed TEFE improves the robustness of lesion feature extraction than single/dual features. The AFFM improves the effectiveness of feature fusion. The proposed method was compared with existing single/multi-modal methods and showed better performance, with an average area under the receiver operating characteristic curve (AUC) of 0.8408 and 0.7913 for prostate cancer classification and Grade Group (GG). Conclusion MAFF-Net uncovers the potential features from BUS, CDFI, and UE images, and provides effective integration of images’ information for enhancing the diagnosis and grading of prostate cancer. The AI network-based multi-modal ultrasonic images aided diagnostic technique presents robustness and clinical applicability in digital pathology.

Accurate tissue classification of chronic prostatitis is critical for pathological research and clinical evaluation. Traditional diagnostic methods are either highly invasive or lack sufficient specificity, making non-invasive and precise quantitative characterization challenging. Photoacoustic imaging (PAI), which enables label-free and high-contrast detection of tissue biochemical properties, provides a novel approach for the non-invasive quantification of prostate inflammation. In this study, 11 inflamed and 9 normal rat prostate tissue sections were used to extract the power spectrum slope of photoacoustic signals at 36 wavelengths, and its efficacy for inflammation classification was evaluated. Results showed the most significant intergroup differences at 720 nm and $770 \mathrm{~nm}(P=0.005)$. The range from 650 to 890 nm contained numerous characteristic wavelengths that effectively distinguished the two tissue types, with negative slopes in the normal group and positive slopes in the inflamed group. This study clarifies the discriminative value of the power spectrum slope parameter, providing reliable quantitative evidence and technical support for the non-invasive detection of prostatitis, with important theoretical and practical significance.

Abstract This study aims to investigate the feasibility of using photoacoustic microscopy for the diagnosis of prostatitis. We induced inflammation in rats to establish a model of prostatitis using Freund’s Complete Adjuvant (FCA). Prostate tissues from both the model and control groups were extracted and processed into histological sections. We explored the photoacoustic microscopy imaging results of unstained sections, consistent with the histological detection results using HE staining. Inflammation was found to enhance the photoacoustic signals. Subsequently, we conducted photoacoustic microscopy imaging on all samples, and the detection results were nearly consistent with the diagnoses made by medical professionals. Finally, we quantified the collected photoacoustic signals to classify the severity of prostatitis.

Elastography is a promising diagnostic tool that measures the hardness of tissues, and it has been used in clinics for detecting lesion progress, such as benign and malignant tumors. However, due to the high cost of examination and limited availability of elastic ultrasound devices, elastography is not widely used in primary medical facilities in rural areas. To address this issue, a deep learning approach called the multiscale elastic image synthesis network (MEIS-Net) was proposed, which utilized the multiscale learning to synthesize elastic images from ultrasound data instead of traditional ultrasound elastography in virtue of elastic deformation. The method integrates multi-scale features of the prostate in an innovative way and enhances the elastic synthesis effect through a fusion module. The module obtains B-mode ultrasound and elastography feature maps, which are used to generate local and global elastic ultrasound images through their correspondence. Finally, the two-channel images are synthesized into output elastic images. To evaluate the approach, quantitative assessments and diagnostic tests were conducted, comparing the results of MEIS-Net with several deep learning-based methods. The experiments showed that MEIS-Net was effective in synthesizing elastic images from B-mode ultrasound data acquired from two different devices, with a structural similarity index of 0.74 ± 0.04. This outperformed other methods such as Pix2Pix (0.69 ± 0.09), CycleGAN (0.11 ± 0.27), and StarGANv2 (0.02 ± 0.01). Furthermore, the diagnostic tests demonstrated that the classification performance of the synthetic elastic image was comparable to that of real elastic images, with only a 3 % decrease in the area under the curve (AUC), indicating the clinical effectiveness of the proposed method.

Objective To enhance the quality of low-resolution (LR) ultrasound images and mitigate artifacts and speckle noise, which can impede accurate medical diagnosis, a novel method called the dual frequency-domain guided adaptation model (DF-GAM) is proposed. The method aims to achieve high-quality image reconstruction across diverse domains, including different ultrasound machines, diseases and phantom images. Methods DF-GAM utilizes a dual-branch network architecture combined with frequency-domain self-adaptation and self-supervised edge regression. This approach enables cross-domain enhancement by focusing on the reconstruction of clear tissue structures and speckle patterns. The model is designed to adapt to various ultrasound imaging (USI) scenarios, ensuring its applicability in real-world clinical settings. Results Experimental evaluations of DF-GAM were conducted using five different datasets. The results demonstrated the method's effectiveness, with DF-GAM outperforming existing enhancement techniques. The average peak signal-to-noise ratio (PSNR) achieved was 34.62, and the structural similarity index (SSIM) was 0.91, indicating a significant improvement in image quality compared to other methods. Conclusion DF-GAM shows great potential in improving medical image diagnosis and interpretation. Its ability to enhance LR ultrasound images across various domains without the need for extensive training data makes it a valuable tool for clinical use. The high PSNR and SSIM scores validate the method's effectiveness, suggesting that DF-GAM could significantly contribute to the field of USI diagnostics.

BACKGROUND: Molecular targeted contrast-enhanced ultrasound (CEUS) imaging is a potential imaging strategy to improve the diagnostic accuracy of conventional ultrasound (US) imaging. US contrast agents are usually micrometer-sized and non-target gas bubbles while nano-sized and targeted agents containing phase-shift materials absorb more attractions for their size and the liquid core and excellent molecular imaging effect. METHODS: PLGA12k-mPEG2k-NH2, DSPE-mPEG2k and perfluorohexan (PFH) were used to construct a new targeted ultrasound contrast agent with CUB domain-containing protein 1 (CDCP1) receptor for the detection and diagnosis of prostate cancer. The potential of tumor-targeted nanoparticles (CDCP1-targeted perfluorohexan-loaded phase-transitional nanoparticles, anti-CDCP1 NPs) as contrast agents for ultrasound (US) imaging was assessed in vitro. Moreover, studies on the cytotoxicity and the targeting ability of anti-CDCP1 NPs assisted by US were carried out. RESULTS: The results showed that anti-CDCP1 NPs had low cytotoxicity, and with the increasing of polymer concentration in anti-CDCP1 NPs, the CEUS imaging of agent gradually enhanced, and enhanced imaging associated with the length of observing time. Furthermore, it was testified that anti-CDCP1 assisted the agent to target cells expressing CDCP1, which demonstrated the active targeting of anti-CDCP1 NPs in vitro. CONCLUSION: All in all, the feasibility of using targeted anti-CDCP1 NPs to enhance ultrasound imaging has been demonstrated in vitro, which laid a solid foundation for molecular US imaging in vivo, and anti-CDCP1 NPs might have a great clinical application prospect.

Background Orthotopic LNCaP xenograft mouse models closely mimic the progression of androgen-dependent prostate cancer in humans; however, orthotopic injection of LNCaP cells into the mouse prostate remains a challenge. Methods Under the guidance of a stereoscopic microscope, the anatomy of the individual prostate lobes in male Balb/c athymic nude mice was investigated, and LNCaP cells were inoculated into the mouse dorsal prostate (DP) to generate orthotopic tumors that mimicked the pathophysiological process of prostate cancer in humans. Real-time ultrasound imaging was used to monitor orthotopic prostate tumorigenesis, contrast-enhanced ultrasonography (CEUS) was used to characterize tumor angiogenesis, and macroscopic and microscopic characteristics of tumors were described. Results The DP had a trigonal bipyramid-shape and were located at the base of the seminal vesicles. After orthotopic inoculation, gray scale ultrasound imaging showed progressive changes in tumor echotexture, shape and location, and tumors tended to protrude into the bladder. After 8 weeks, the tumor take rate was 65% ( n = 13/20 mice). On CEUS, signal intensity increased rapidly, peaked, and decreased gradually. Observations of gross specimens showed orthotopic prostate tumors were well circumscribed, round, dark brown, and soft, with a smooth outer surface and a glossy appearance. Microscopically, tumor cells were arranged in acini encircled by fibrous septa with variably thickened walls, mimicking human adenocarcinoma. Conclusions This study describes a successful approach to establishing an orthotopic LNCaP xenograft Balb/c athymic nude mouse model. The model requires a thorough understanding of mouse prostate anatomy and proper technique. The model represents a valuable tool for the in vivo study of the biological processes involved in angiogenesis in prostate cancer and preclinical evaluations of novel anti-angiogenic therapies.

BACKGROUND:Understanding angiogenesis in prostate cancer is essential. LNCaP prostate xenograft tumors are androgen responsive and closely mimic clinical disease. Orthotopic animal models replicate aspects of the cancer microenvironment and are more clinically relevant than subcutaneous models. Comparative studies investigating angiogenesis using contrast-enhanced ultrasound (CEUS) imaging in subcutaneous and orthotopic mouse models of prostate cancer have not been performed.METHODS:Tumor microcirculation and perfusion in subcutaneous and orthotopic LNCaP xenograft Balb/c athymic nude mice models were compared by investigating microbubble wash-in with CEUS.RESULTS:The take rate of subcutaneous and orthotopic tumors were 58.3% and 68.2%, respectively. On CEUS, orthotopic prostate tumors enhanced more rapidly than subcutaneous tumors. Mean arrival-time (Atm) for subcutaneous tumors, orthotopic prostate tumors, and kidney were 4.21±1.86, 1.72±0.79, and 0.73±0.12 s, respectively. Mean Atm was significantly longer for subcutaneous tumors compared to orthotopic prostate tumors or kidney (P<0.01). Mean time to peak enhancement (TtoPk) for subcutaneous tumors, orthotopic prostate tumors, and kidney were 38.56±13.23, 12.39±7.17, and 3.74±1.41 s, respectively. Mean TtoPk were significantly shorter for orthotopic prostate tumors and kidney compared to subcutaneous tumors (P<0.01). Mean wash-in area under the curve (WiAuC) for subcutaneous tumors, orthotopic prostate tumors, and kidney were 611.11±247.52, 1,800.57±623.11, and 1,887.51±103.68 dB, respectively. Mean AUC was significantly higher for orthotopic prostate tumors and kidney compared to subcutaneous tumors (P<0.01). Parametric imaging confirmed these findings. The density of CD31-positive vessels was significantly higher in orthotopic prostate tumors (43.98±6.14 vessels/field) compared to subcutaneous tumors (15.44±3.74 vessels/field, P<0.01).CONCLUSIONS:These findings demonstrate that orthotopic LNCaP xenografts better recreate a pro-angiogenic microenvironment than subcutaneous LNCaP xenografts.

In the 1980's scurvy is rare. The majority of patients are at the two extremes of age, being either young children or elderly persons, who have faulty diets of milk products without fresh fruits or vegetables. Such a case, a 2.5 year-old boy, is presented and discussed. Blood dyscrasias, anaemias and osteoporosis should be considered in the differential diagnosis.

Objective:To explore the diagnostic value of transrectal ultrasound(TRUS)/multiparametric magnetic resonance imaging(mpMRI) fusion targeted biopsy(FTB) for clinically significant prostate cancer(PCa) detection by using both biopsy histopathology and radical prostatectomy histopathology as reference standards.Methods:A total of 303 consecutive patients with suspicious lesions detected by mpMBI and underwent prostate biopsy at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine between November 2017 to January 2020 were retrospectively analyzed. All the suspicious lesions were sampled by TRUS/mpMRI FTB in addition with standard 12-core systematic biopsy(SB). The clinically significant PCa detection rates by TRUS/mpMRI FTB and SB were compared by using both biopsy histopathology and radical prostatectomy histopathology as reference standards.Results:The diagnosis of PCa was histologically confirmed in 189 of 303 patients, including 178 patients with clinically significant PCa and 11 patients with clinically insignificant PCa. With biopsy histopathology as reference standard, the clinically significant PCa detection rate of TRUS/mpMRI FTB was statistically higher than SB (57.1% vs 45.9%, P<0.001). Among 189 patients with biopsy proven PCa, 80 patients underwent radical prostatectomy, and the radical prostatectomy histopathology confirmed 79 patients with clinically significant PCa.With radical prostatectomy as reference standard, the clinically significant PCa detection rate of TRUS/mpMRI FTB was statistically higher than SB (91.1% vs 74.7%, P<0.001). Conclusions:Compared with SB, MRI/US FTB can offer more accurate sampling of suspicious lesions on mpMRI, and consequently improve the clinically significant PCa detection rate.

NanomedicineVol. 16, No. 23 EditorialUltrasound-based nanomedicine for molecular imaging of prostate cancer: from diagnostics to theranosticsYunkai Zhu‡, Ying Sun‡, Yourong Duan & Yaqing ChenYunkai Zhu‡ https://orcid.org/0000-0002-5457-7994Department of Ultrasound in Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai, 200092, PR China, Ying Sun‡State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200032, PR China, Yourong Duan *Author for correspondence: E-mail Address: yrduan@shsci.orghttps://orcid.org/0000-0002-3781-7845State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200032, PR China & Yaqing Chen **Author for correspondence: E-mail Address: chenyaqing@xinhuamed.com.cnhttps://orcid.org/0000-0001-5132-0096Department of Ultrasound in Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai, 200092, PR ChinaPublished Online:14 Sep 2021https://doi.org/10.2217/nnm-2021-0170AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: drug deliverynanobubblenanodropletnanomedicineprostate cancertheranosticsultrasoundReferences1. 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Theranostics 8(6), 1665–1677 (2018).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 16, No. 23 Follow us on social media for the latest updates Metrics Downloaded 81 times History Received 30 April 2021 Accepted 11 August 2021 Published online 14 September 2021 Published in print October 2021 Information© 2021 Future Medicine LtdKeywordsdrug deliverynanobubblenanodropletnanomedicineprostate cancertheranosticsultrasoundFinancial & competing interests disclosureThis project was supported by grants from the National Natural Science Foundation of China (no. 81671708, 81271595, 81572999, 81771839 and 81773272), Shanghai Shen Kang Hospital Development Center Research Project (no. 16CR3092B) and the Fund of Shanghai Jiaotong University (no. YG2014ZD04). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

目的:探讨超声造影前后能否提高甲状腺影像报告和数据系统(thyroid image report and data system,TI-RADS)诊断效能.方法:选择2017年12月至2019年5月行甲状腺超声结节造影的患者151例,共152个结节,分析152个结节造影前后的二维声像特征差异,比较造影前后TI-RADS分级诊断、造影过程联合造影后TI-RADS的诊断效能.结果:造影前及造影后二维声像图上甲状腺结节边缘、钙化有统计学差异(P<0.05),回声类型无统计学差异(P>0.05).造影前后TI-RADS分级诊断的敏感性、特异性、准确率分别为83.51％、45.45％、69.73％和96.91％、40.00％、76.32％.造影过程联合造影后TI-RADS分级的敏感性、特异性、准确率分别为83.51％、63.64％、76.32％.造影后TI-RADS、造影过程联合造影后TI-RADS的受试者工作特征曲线下面积均高于造影前(P<0.05).结论:超声造影技术能提高二维声像图甲状腺结节边缘、钙化特征判读的准确性,造影后TI-RADS分级具有更高的敏感性,造影过程联合造影后TI-RADS具有更高的特异性,超声造影技术能够提高TI-RADS分级的诊断效能.

Implementing prostate biopsy with the guidance of magnetic resonance imaging (MRI) can reduce the risk and improve the accuracy in prostate cancer therapy. How to combine information from MRI and transrectal ultrasonography (TRUS) plays an important role in image fusion-guided biopsy. In this study, we introduce a neural network based on U-net for prostate segmentation in MRI and TRUS. On the basis of the contour generated from segmentation masks, we implement the thin plate spline-robust point matching for non-rigid registration. We validate our method using animal experiments with simulated lesions and obtain convincing results. The results show that our model achieve a mean intersection over union (mIOU) of 0.819 for TRUS segmentation and a mIOU of 0.878 for MRI segmentation. We also modify root mean square error (RMSE) and achieve the result of 0.0082 in registration which is more accurate than rigid registration.

目的:探讨超声引导下聚桂醇与射频消融治疗囊性为主甲状腺结节的有效性.方法:选取2015年2月—2018年5月上海中医药大学附属曙光医院收治的囊性为主甲状腺结节患者76例,依据治疗方法不同,分为聚桂醇组(N=51)和射频组(N=25).术后观测两组患者的临床疗效及不良反应发生率.结果:术后12个月射频组总有效率为100.00％(25/25),高于聚桂醇组总有效率[84.31％(43/51)],差异有统计学意义(P<0.05);聚桂醇组不良反应率为13.73％(7/51),射频组不良反应率为16.00％(4/25),两组不良反应率差异无统计学意义(P>0.05).结论:超声引导下射频消融治疗对囊性为主的甲状腺结节疗效更显著,聚桂醇治疗具有经济优势,二种方法均值得临床推广.

Objective:To assess the utility of contrast-enhanced ultrasound (CEUS) targeted biopsy (TB) for clinically significant prostate cancer (PCa) detection.Methods:A total of 983 consecutive patients scheduled for prostate biopsy from October 2015 to March 2019 in Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine were enrolled in this retrospective study. All patients had suspicious lesions on CEUS, defined as increased focal contrast enhancement, rapid contrast enhancement and low enhancement lesions with ill-defined borders. Suspicious lesions on CEUS were sampled in addition with standard 12-core systematic biopsy(SB). Clinically significant PCa was defined using Epstein criteria. The clinically significant PCa detection rate by CEUS-TB and combined biopsy was evaluated in comparison with SB.Results:In 502 of the 983 patients, the diagnosis of PCa was histologically confirmed, including 445 patients with clinically significant PCa and 57 patients with clinically insignificant PCa. The clinically significant PCa by CEUS-TB and combined biopsy were 41.9% (412/983) and 45.3% (445/983) respectively, which was significantly higher than SB (36.8%, 362/983)(all P<0.001). CEUS-TB resulted in additional 83 cases of clinically significant PCa, including 61 patients missed by SB and 22 patients under-graded by SB. Conclusions:CEUS is helpful in the detection of PCa lesions. Combined CEUS-TB and SB can improve the clinically significant PCa detection rate.

Aim: To design MRI/ultrasound (US) dual modality imaging probes with optimized size for prostate cancer imaging by targeting prostate-specific membrane antigen (PSMA). Materials & methods: The PSMA-targeting polypeptide-nanobubbles (PP-NBs) with core size of 400 and 700 nm were fabricated and evaluated. Results: With excellent physical property and specificity, PP-NBs of both core size could image PSMA expression in prostate cancer xenografts. Particularly, 400 nm PP-NBs generated higher PSMA-specific MRI/US dual modality contrast enhancement than 700 nm PP-NBs in correlation with histopathologic findings. Conclusion: Benefit from the smaller core size, 400 nm PP-NBs had higher permeability and specificity than 700 nm PP-NBs, hence producing better PSMA-specific MRI/US dual modality imaging.

Objective To retrospectively investigate the value of contrast enhanced ultrasound ( CEUS) in breast cancer biopsy . Methods A total of 49 consecutive patients with biopsy confirmed breast cancer were retrospectively analyzed . All patients underwent CEUS and biopsies were thus performed targeting both the high perfusion and low/non‐perfusion regions on CEUS . T he diagnostic performance and core cancer involvement of the biopsy cores taken from the high perfusion regions were compared with those from the low/non‐perfusion . Results A total of 53 breast cancer lesions were biopsy confirmed in 49 patients .CEUS revealed homogeneous enhancement in 8 lesions ( 15 .1% ) ,and heterogeneous enhancement in 45 lesions ( 84 .9% ) . T he diagnostic accuracy rate for biopsy cores taken from the high perfusion regions was significantly higher than that from the low/non‐perfusion regions ( 98 .5% vs 72 .9% , P ＜0 .01) . T he core cancer involvement was also higher in high perfusion lesions ( 55% vs 30% , P ＜0 .01) . Conclusions CEUS can differentiate the active area and necrotic fibrosis area of breast tumors by displaying the microvessels ,thus contributing to the selection of biopsy sites .

Objective To investigate prostate cancer detection rate of different biopsy protocols in different PSA value groups in rural China. Methods A total of 186 patients underwent contrast-enhanced ultrasound (CEUS) in order to determine the puncture target prior to biopsy were enrolled in this retrospective study. All patients underwent 12-core SB combined with CEUS-TB. The biopsy results of different biopsy protocols were compared in patients with stratification by PSA value. Results In 186 patients underwent prostate biopsy, the histopathologic results revealed prostate cancer in 117 cases (62.9%) and benign lesions in 69 cases (37.1%). The PSA 4-10ng/ml group, the prostate cancer detection rate of 8-core and 12-core method was no statistical significance. But the 12-core method was significantly higher than CEUS-TB (44.9% versus 32.7%, P = 0.01). The PSA 10-20ng/ml group, there was no statistical significance in prostate cancer detection rate between SB and CEUS-TB (50.0% versus 45.7%, P = 0.15). The PSA greater than 20ng/ml group, the prostate cancer detection rate by SB was higher than CEUS targeted biopsy, but there was no statistical significance (79.1% versus 76.9%, P = 0.15). In the overall patients, the biopsy core positive rate of CEUS-TB was significantly higher than SB (97% versus 55.5% and 28.5%, P = 0.0001). Conclusion The flexible use of SB combined with CEUS-TB can reduce the number of biopsy cores in higher PSA groups. It has clinical importance in the detection of prostate cancer in different PSA value groups in rural china.