Jasmonate inhibits plant growth and reduces gibberellin levels via microRNA5998 and transcription factor MYC2.

Jasmonate (JA) and gibberellins (GAs) exert antagonistic effects on plant growth and development in response to environmental and endogenous stimuli. Although the crosstalk between JA and GA has been elucidated, the role of JA in GA biosynthesis remains unclear. Therefore, in this study, we investigated the mechanism underlying JA-mediated regulation of endogenous GA levels in Arabidopsis (Arabidopsis thaliana). Transient and electrophoretic mobility shift assays showed that transcription factor MYC2 regulates GA inactivation genes. Using transgenic plants, we further evaluated the contribution of MYC2 in regulating GA inactivation genes. JA treatment increased DELLA accumulation but did not inhibit DELLA protein degradation. Additionally, JA treatment decreased bioactive GA content, including GA4, significantly decreased the expression of GA biosynthesis genes, including ent-kaurene synthase (AtKS), GA 3ß-hydroxylase (AtGA3ox1), and AtGA3ox2, and increased the expression of GA inactivation genes, including GA 2 oxidase (AtGA2ox4), AtGA2ox7, and AtGA2ox9. Conversely, JA treatment did not significantly affect gene expression in the myc2 myc3 myc4 triple mutant, demonstrating the MYC2-4-dependent effects of JA in GA biosynthesis. Additionally, JA post-transcriptionally regulated AtGA3ox1 expression. We identified microRNA miR5998 as an AtGA3ox1-associated miRNA; its overexpression inhibited plant growth by suppressing AtGA3ox1 expression. Overall, our findings indicate that JA treatment inhibits endogenous GA levels and plant growth by decreasing the expression of GA biosynthesis genes and increasing the expression of GA inactivation genes via miR5998 and MYC2 activities.

Association of left pulmonary vein trunk and subclinical atrial fibrillation in patients with cardiac implantable electronic device.

The relationship between subclinical atrial fibrillation (SCAF) and left pulmonary vein anatomy is unknown. This study sought to investigate whether left pulmonary vein trunk predict the development of SCAF in patients with cardiac implantable electronic device (CIED). We also examined the relationship between the duration of SCAF and left pulmonary vein trunk. We retrospectively enrolled 162 patients who underwent implantation of dual-chamber CIEDs and follow-up by remote monitoring system. Computed tomography was used to measure the length of the left pulmonary vein. During median follow up of 2.7 years, the episodes of > 6 min and > 24 h SCAF were observed in 61 (37.7%) and 24 (14.8%) patients, respectively. The diagnosis of sinus node disease (HR: 3.66 [2.06-6.52], P < 0.01 and HR: 2.68 [1.09-6.62], P = 0.04) and left atrial diameter (HR: 1.04 [1.00-1.07], P = 0.04 and HR: 1.05 [1.00-1.10], P = 0.04) were independent predictors for > 6 min and > 24 h SCAF, respectively. Length of the left pulmonary vein trunk was an independent predictor for > 6 min SCAF (HR: 1.06 [1.02-1.10], P < 0.01), but not for > 24 h SCAF (P = 0.06). Sinus node disease, size of the left atrium and length of the left pulmonary vein trunk were related to SCAF. The left pulmonary vein trunk might especially contribute as a trigger rather than as a driver of development of atrial fibrillation.

DELLA-GAF1 complex is involved in tissue-specific expression and gibberellin feedback regulation of GA20ox1 in Arabidopsis.

KEY MESSAGE: The GAF1 transcription factor is shown to bind to the promoter of the Arabidopsis GA-biosynthetic enzyme GA20ox1 and, in association with DELLA protein, promotes GA20ox1 expression, thereby contributing to its feedback regulation and tissue specificity. Gibberellins (GAs) are phytohormones that promote plant growth and development, including germination, elongation, flowering, and floral development. Homeostasis of endogenous GA levels is controlled by GA feedback regulation. DELLAs are negative regulators of GA signaling that are rapidly degraded in the presence of GAs. DELLAs regulate several target genes, including AtGA20ox2 and AtGA3ox1, encoding the GA-biosynthetic enzymes GA 20-oxidase and GA 3-oxidase, respectively. Previous studies have identified GAI-ASSOCIATED FACTOR 1 (GAF1) as a DELLA interactor, with which DELLAs act as transcriptional coactivators; furthermore, AtGA20ox2, AtGA3ox1, and AtGID1b were identified as target genes of the DELLA-GAF1 complex. Among the five Arabidopsis GA20ox genes, AtGA20ox1 is the most highly expressed gene during vegetative growth; its expression is controlled by GA feedback regulation. Here, we investigated whether AtGA20ox1 is regulated by the DELLA-GAF1 complex. The electrophoretic mobility shift and transactivation assays showed that three GAF1-binding sites exist in the AtGA20ox1 promoter. Using transgenic plants, we further evaluated the contribution of the DELLA-GAF1 complex to GA feedback regulation and tissue-specific expression. Mutations in two GAF1-binding sites obliterated the negative feedback regulation and tissue-specific expression of AtGA20ox1 in transgenic plants. Thus, our results showed that GAF1-binding sites are involved in GA feedback regulation and tissue-specific expression of AtGA20ox1 in Arabidopsis, suggesting that the DELLA-GAF1 complex is involved in both processes.

A Stacked Back Side-Illuminated Voltage Domain Global Shutter CMOS Image Sensor with a 4.0 µm Multiple Gain Readout Pixel †.

A backside-illuminated complementary metal-oxide-semiconductor (CMOS) image sensor with 4.0 µm voltage domain global shutter (GS) pixels has been fabricated in a 45 nm/65 nm stacked CMOS process as a proof-of-concept vehicle. The pixel components for the photon-to-voltage conversion are formed on the top substrate (the first layer). Each voltage signal from the first layer pixel is stored in the sample-and-hold capacitors on the bottom substrate (the second layer) via micro-bump interconnection to achieve a voltage domain GS function. The two sets of voltage domain storage capacitor per pixel enable a multiple gain readout to realize single exposure high dynamic range (SEHDR) in the GS operation. As a result, an 80dB SEHDR GS operation without rolling shutter distortions and motion artifacts has been achieved. Additionally, less than -140dB parasitic light sensitivity, small noise floor, high sensitivity and good angular response have been achieved.

A 120-ke- Full-Well Capacity 160-µV/e- Conversion Gain 2.8-µm Backside-Illuminated Pixel with a Lateral Overflow Integration Capacitor.

In this paper, a prototype complementary metal-oxide-semiconductor (CMOS) image sensor with a 2.8-µm backside-illuminated (BSI) pixel with a lateral overflow integration capacitor (LOFIC) architecture is presented. The pixel was capable of a high conversion gain readout with 160 µV/e- for low light signals while a large full-well capacity of 120 ke- was obtained for high light signals. The combination of LOFIC and the BSI technology allowed for high optical performance without degradation caused by extra devices for the LOFIC structure. The sensor realized a 70% peak quantum efficiency with a normal (no anti-reflection coating) cover glass and a 91% angular response at ±20° incident light. This 2.8-µm pixel is potentially capable of higher than 100 dB dynamic range imaging in a pure single exposure operation.

Combination of Lesion Stenosis and Myocardial Supply Area Assessed by Coronary Computed Tomography Angiography for Prediction of Myocardial Ischemia.

Recent clinical studies revealed that anatomical information assessed by coronary computed tomography angiography (CTA) may be used effectively to diagnose coronary artery disease (CAD). However, a physiological assessment, demonstrating myocardial ischemia, is required to justify a therapeutic strategy for CAD. This study aimed to investigate whether using CTA to assess myocardial supply area can improve the prediction of myocardial ischemia.We analyzed 201 vessels with moderate (luminal narrowing ≥ 50%, < 70%) and severe (luminal narrowing ≥ 70%, < 99%) stenosis on CTA from 174 patients, who were suspected of having stable angina and underwent measurement of fractional flow reserve (FFR). The myocardial area supplied by the coronary artery, distal to the stenosis, was evaluated with CTA, as reported previously (modified Alberta Provincial Project for Outcome Assessment in Coronary Heart score) and was classified into 3 groups (large, medium, and small).Both percentage area stenosis and myocardial supply area were significantly correlated with FFR (r = -0.46, P < 0.01, and r = -0.45, P < 0.01). Among patients who had coronary plaques, with moderate stenosis and a small myocardial supply area, only 3 of 42 lesions (7%) were identified as ischemic; deviation from the ischemic threshold (FFR = 0.80) was P < 0.01. The combined assessment of lesion stenosis and myocardial supply area, using CTA, improved the prediction of myocardial ischemia significantly compared to lesion stenosis alone (77% versus 59%, P < 0.01).Adding the assessment of myocardial supply area to standard CTA might help predict myocardial ischemia in patients with stable angina pectoris.

Retrospective Comparison of Long-Term Clinical Outcomes Between Percutaneous Coronary Intervention and Medical Therapy in Stable Coronary Artery Disease With Gray Zone Fractional Flow Reserve　- COMFORTABLE Retrospective Study.

BACKGROUND: A fractional flow reserve (FFR) between 0.75 and 0.80 constitutes a "gray zone" for clinical decision-making in coronary artery disease. We compared long-term outcomes of percutaneous coronary intervention (PCI) using drug-eluting stents vs. medical therapy for coronary stenosis with gray zone FFR. Methods and Results: We retrospectively investigated the clinical outcomes of 263 patients with gray zone FFR: 78 patients in the PCI group and 185 patients in the medical therapy group. During a median follow-up of 3.7 years, the frequency of target vessel failure (TVF, defined as a composite of cardiac death, myocardial infarction [MI], or ischemia-driven target vessel revascularization [TVR]) was significantly lower in the PCI group compared with the medical therapy group (6% vs. 19%, hazard ratio [HR]:0.33, 95% confidence interval [CI]: 0.13-0.84, P=0.008). The frequency of a composite of cardiac death or MI was not different between the 2 groups (1% vs. 2%, HR: 0.61, 95% CI: 0.07-5.49, P=0.645). The frequency of ischemia-driven TVR was significantly lower in the PCI group compared with the medical therapy group (5% vs. 18%, HR: 0.28, 95% CI: 0.10-0.79, P=0.005). CONCLUSIONS: In patients with gray zone FFR, compared with medical therapy, PCI decreased the frequency of TVF, which was mainly driven by a reduction in the frequency of angina or myocardial ischemia without any difference in the frequency of cardiac death or MI.

An Over 90 dB Intra-Scene Single-Exposure Dynamic Range CMOS Image Sensor Using a 3.0 µm Triple-Gain Pixel Fabricated in a Standard BSI Process.

To respond to the high demand for high dynamic range imaging suitable for moving objects with few artifacts, we have developed a single-exposure dynamic range image sensor by introducing a triple-gain pixel and a low noise dual-gain readout circuit. The developed 3 µm pixel is capable of having three conversion gains. Introducing a new split-pinned photodiode structure, linear full well reaches 40 ke-. Readout noise under the highest pixel gain condition is 1 e- with a low noise readout circuit. Merging two signals, one with high pixel gain and high analog gain, and the other with low pixel gain and low analog gain, a single exposure dynamic rage (SEHDR) signal is obtained. Using this technology, a 1/2.7", 2M-pixel CMOS image sensor has been developed and characterized. The image sensor also employs an on-chip linearization function, yielding a 16-bit linear signal at 60 fps, and an intra-scene dynamic range of higher than 90 dB was successfully demonstrated. This SEHDR approach inherently mitigates the artifacts from moving objects or time-varying light sources that can appear in the multiple exposure high dynamic range (MEHDR) approach.

Role of the 5-HT1A autoreceptor in the enhancement of fluvoxamine-induced increases in prefrontal dopamine release by adrenalectomy/castration in mice.

We have found that fluvoxamine-induced increases in prefrontal dopamine release are enhanced by adrenalectomy/castration and 5-HT1A receptors are involved in the enhancement. This study examined which 5-HT1A autoreceptors or postsynaptic receptor play a key role in the enhancement in mice. Adrenalectomy/castration-induced enhancement of fluvoxamine-induced increase in the dopamine release was not blocked by local perfusion with the 5-HT1A receptor antagonist WAY100635 (10 µM), while it was blocked by systemic administration of WAY100635 at low dose (0.1 mg/kg) which blocked preferentially autoreceptor-mediated responses. These finding suggests that 5-HT1A autoreceptors play a key role in the enhancement of prefrontal dopamine release.

[Development of Auto Dense-breast Classification on Mammography Images Using Image Similarity].

PURPOSE: In Japan, radiologists perform qualitative visual classification to define four categories of mammary gland density. However, an objective estimation of mammary gland density is necessary. To address this, we developed an automatic classification software using image similarity. METHODS: We prepared 741 cases of mediolateral oblique images (MLO) for evaluation, and they were diagnosed as normal among the mammography images taken at our hospital. Image matching was performed using the evaluation images and an image database for breast density determination. In this study, the image similarity used zero normalized cross-correlation (ZNCC) as an index. In addition, if the breast thickness is less than 30 mm and each breast density category ZNNC has the same value, the category is evaluated on the fat side. We compared the results of qualitative visual classification and automatic classification methods to assess consistency. RESULTS: The agreement with the subjective breast composition classification was 78.5%, and the weighted kappa coefficient was 0.98. One mismatched case was evaluated on the higher density side with the same ZNCC value between categories and a breast thickness greater than 30 mm. CONCLUSION: Image similarity provides an excellent estimation of quantification of breast density. This system could contribute to improving the efficiency of the mammography screening system.

Patient radiation exposure dose reduction using stent-enhanced image processing in percutaneous coronary intervention.

We investigated the reduction in patient radiation exposure dose during percutaneous coronary intervention (PCI) by stent enhancement processing. We examined the effects of dose reduction based on the image quality of stent enhancement processing using a purpose-built dynamic phantom. We evaluated the image contrast (IC) of the stent in stent-enhanced images (SVref), digital angiography (DA), and stent-enhanced images with a 20%, 40%, and 60% lower imaging doses (SV20, SV40, and SV60). We visually evaluated graininess and stent shape using the mean opinion score (MOS) and retrospectively evaluated the acquisition duration of stent enhancement in PCI cases; finally, we estimated the decrease in patient radiation exposure due to stent enhancement. The image contrast of SVref at phantom thicknesses of 20 cm was 51.25 ± 3.82, while the image contrast of DA was significantly reduced at 14.90 ± 1.57 (p < 0.05). We observed a significant decrease in the MOS of graininess in SV60 and MOS of stent shape in DA (p < 0.05). Furthermore, the average imaging duration for stent enhancement using PCI was 22.65 ± 7.43 s, and the maximum imaging duration was 68.07 s. We hypothesize that patient radiation exposure dose can be reduced by up to 60.17 mGy by lowering the imaging dose during the stent enhancement process. Stent enhancement processing improves the visibility of stent images, and can reduce radiation exposure by approximately 40% during confirmation imaging of stents. Our study contributes to the reduction of radiation exposure dose for operators and patients in PCI.

Impact of tag index and local electrogram for successful first-pass cavotricuspid isthmus ablation.

Background: The optimal ablation index (AI) value for cavotricuspid isthmus (CTI) ablation is unknow. Objective: This study investigated the optimal AI value and whether preassessment of local electrogram voltage of CTI could predict first-pass success of ablation. Methods: Voltage maps of CTI were created before ablation. In the preliminary group, the procedure was performed in 50 patients targeting an AI ≥450 on the anterior side (two-thirds segment of CTI) and AI ≥400 on the posterior side (one-third segment of CTI). The modified group also included 50 patients, but the target AI for the anterior side was modified to ≥500. Results: In the modified group, the first-pass rate of success was higher (88% vs 62%; P < .01) than in the preliminary group, and there were no differences in the average bipolar and unipolar voltages at the CTI line. Multivariate logistic regression analysis revealed that ablation with an AI ≥500 on the anterior side was the only independent predictor (odds ratio 4.17; 95% confidence interval 1.44-12.05; P < .01). The bipolar and unipolar voltages were higher at sites without conduction block than at sites with conduction block (both P < .01). The cutoff values for predicting conduction gap were ≥1.94 mV and ≥2.33 mV with areas under the curve of 0.655 and 0.679, respectively. Conclusions: CTI ablation with a target AI >500 on the anterior side was shown to be more effective than an AI >450, and local voltage at a conduction gap was higher than without a conduction gap.

[Development and Validation of Dose Management Software in IVR Utilizing Radiation Information System].

PURPOSE: In each clinical case, the equipment display dose is used for interventional radiology (IVR) dose management and conform to the Japan diagnostic reference levels 2020 (DRLs 2020). However, dose management software corresponding to the DRLs 2020 using radiation dose structured report (RDSR) is not sufficiently widespread. This study aimed to assess the usefulness of in-house developed dose management software in IVR utilizing radiology information system (RIS), which can record both procedures and lesions. METHODS: In this study, IVR from July to September 2020 was analyzed (cardiac regions: 141 cases and other regions: 149 cases). The evaluation items were air kerma-area product, air kerma at the patient entrance reference point, and patient information (height, weight, and BMI). Each subject of DRLs 2020 was analyzed by 12 radiological technologists in charge of IVR. The difference between results of the manual analysis and those of the in-house developed dose management software was calculated using paired t-test in terms of analysis time. RESULTS: The analysis time for the cardiac and other regions was 4180.25±1161.79 s and 4366.92±1393.19 s in the manual analysis, and 36.25±15.32 s and 38.08±17.34 s in the software. The use of software indicated a significant reduction in analysis time (p<0.05). The analysis accuracy of the cardiac and other regions was 96.30% and 98.89% in the software. CONCLUSION: These results show the usefulness of dose management software utilizing RIS.

Clinical Features of Glaucoma Associated with Cytomegalovirus Corneal Endotheliitis.

Purpose: The purpose of this study was to highlight the manifestations of glaucoma associated with cytomegalovirus (CMV) corneal endotheliitis. Methods: We reviewed the 34 patients that met the diagnostic criteria for CMV endotheliitis in our hospital, with special attention to the glaucoma status, including onset of glaucoma, glaucoma in the fellow eye, visual field defects, intraocular pressure, and final outcomes. Results: Thirty-four eyes of 34 patients (mean age, 69.1 ± 13.1 years; 31 males [91.2%]) with CMV corneal endotheliitis were enrolled. Thirty-two eyes (94.1%) had a history of a glaucoma diagnosis, which had been treated for 10.0 ± 10.1 years. Glaucoma in the fellow eye was noted in 16 cases (47.1%) and a history of Posner-Schlossman syndrome was noted in 13 cases (38.2%). Visual fields measured using a Humphrey field analyzer were normal-to-early stage (MD>-6dB) in 16 eyes (47.1%) and middle-to-late stage (MD≤-6dB) in 18 eyes (52.9%). The intraocular pressure decreased from 22.4 ± 10.6 mmHg at the initial visit to 14.9 ± 7.9 mmHg after medical treatment, including 0.5% topical ganciclovir (GCV) with and without a systemic anti-CMV agent, corticosteroid eye drops, and an anti-glaucoma agent (p<0.01). During the follow-up period of 4.8 ± 3.0 years (range, 0.2-10 years), 16 eyes (47.1%) required glaucoma surgery, including filtering surgery (7 eyes) and trabeculotomy only (9 eyes). Conclusion: Our case series showed that most of the patients with CMV corneal endotheliitis had glaucoma. Although medical therapy, including 0.5% topical GCV, had efficacy in lowering the intraocular pressure, one-half of the cases required glaucoma surgery. Therefore, ophthalmologists should strive to make an earlier diagnosis of CMV corneal endotheliitis by utilizing PCR testing of aqueous humor samples to prevent sight-threatening glaucomatous damage.

Effect of manuka honey on human immunodeficiency virus type 1 reverse transcriptase activity.

Manuka honey (MkH), derived from New Zealand manuka tree (Leptospermum scoparium), is considered a therapeutic agent owing to its antibacterial, antioxidant, antifungal, antiviral, anti-inflammatory, and wound healing activities. In this study, the inhibitory effect of five honey types, including MkH, on HIV-1 RT activity was evaluated, using an RT assay colorimetric kit, according to the manufacturer's instructions with slight modifications. MkH exerted the strongest inhibitory effect in a dose-dependent manner, with a half maximal inhibitory concentration (IC50) of approximately 14.8 mg/mL. Moreover, among the MkH constituents, methylglyoxal (MGO) and 2-methoxybenzoic acid (2-MBA) were determined to possess anti-HIV-1 RT activity. MGO and 2-MBA in MkH were identified by High Performance Liquid Chromatography (HPLC) and Liquid Chromatograph - Mass Spectrometry (LC-MS/MS). The findings suggest that the inhibitory effect of MkH on the HIV-1 RT activity is mediated by multiple constituents with different physical and chemical properties.

One-color reversible control of photochromic reactions in a diarylethene derivative: three-photon cyclization and two-photon cycloreversion by a near-infrared femtosecond laser pulse at 1.28 µm.

One-color control of colorization/decolorization reactions of diarylethene molecules was attained by using nonresonant high-order multiphoton absorption processes with a near-infrared (NIR) femtosecond laser pulse at 1.28 µm with 35 fs full width at half-maximum (fwhm). The intensity of a rather weak laser pulse (<1 nJ/pulse) can induce the simultaneous three-photon absorption leading to the colorization, while much weaker intensity induces two-photon absorption resulting in the decolorization. The spatial patterning concomitant with higher-order multiphoton absorption processes was also demonstrated.

Foxn1 is essential for vascularization of the murine thymus anlage.

We addressed whether vascularization of the thymus anlage depends on Foxn1 expression. In the thymus anlagen of wild-type mice, CD31(+) endothelial cells are initially observed between epithelial cells on embryonic day (Ed)12.5 and form luminal structure on Ed13. VEGF are produced in epithelial cells and mesenchymal cells which invaginate in the epithelial region of the anlagen on Ed13. However, in the nude thymus anlagen, neither CD31(+) cells nor VEGF producing mesenchymal cells is detected in the epithelial region. The present results indicate that Foxn1 dependent epithelial development is essential for vascularization of the thymus anlagen.

Pharmacological profile of the novel anti-inflammatory corticosteroid NS-126, a therapeutic agent for allergic rhinitis.

NS-126 (9-fluoro-11beta,17,21-trihydroxy-16alpha-methylpregna-1,4-diene-3,20-dione 21-cyclohexanecarboxylate 17-cyclopropanecarboxylate) is a novel, highly lipophilic anti-inflammatory corticosteroid. We compared NS-126 and the widely used intranasal corticosteroid fluticasone propionate (FP) in a guinea-pig model of allergic rhinitis and a rat model of airway eosinophilia. In the allergic rhinitis model, NS-126 and FP reduced sneezing and nasal obstruction to similar extents. In the airway eosinophilia model, both compounds inhibited the infiltration of eosinophils into the bronchoalveolar lavage fluid, but the effect of NS-126 was longer-lasting than that of FP. In vitro, NS-126 showed lower affinity than FP for the glucocorticoid receptor and was a weaker inhibitor of Th(2) cytokine and chemokine production and mast-cell secretory responses. We also investigated DX-17-CPC, a metabolite of NS-126 generated in nasal tissue by carboxylesterase-catalyzed hydrolysis at the 17-position. DX-17-CPC showed greater affinity than NS-126 for the glucocorticoid receptor and was a stronger inhibitor of Th(2) cytokine and chemokine production and mast-cell secretory responses. The long duration of the anti-allergic effects of NS-126 may be explained by its high lipophilicity, while the strength of its anti-allergic effects may be explained by the generation of the active metabolite DX-17-CPC. NS-126 is a long-acting intranasal corticosteroid and a promising therapeutic agent for allergic rhinitis.

Involvement of the NADPH oxidase 2 pathway in renal oxidative stress in Aqp11 -/- mice.

Aquaporin-11 (AQP11) is an intracellular AQP. Several studies with Aqp11 -/- mice have shown that AQP11 has a role in normal development of the kidney after birth. Our previous studies have suggested that alteration of oxygen homeostasis may be involved in the kidney injury caused by AQP11 deficiency, although the underlying mechanism is largely unknown. To clarify this issue, we examined genes that are related to oxygen homeostasis in Aqp11 -/- mice. Among 62 genes that are involved in oxygen homeostasis, 35 were upregulated by more than 2-fold in Aqp11 -/- mice in comparison with wild-type mice. Pathway analysis using these genes extracted the pathway responsible for production of reactive oxygen species in macrophages. As expression of the genes involved in the NADPH oxidase 2 (NOX2) complex was dramatically increased by more than 14-fold, we further analyzed NOX2 at the protein level. Immunoblotting analysis demonstrated a dramatic increase of NOX2 protein in the kidney of Aqp11 -/- mice, and immunohistochemistry showed that NOX2 protein and a marker protein for macrophages were increased in the renal interstitium. These results indicate that NOX2-induced oxidative stress accompanied by macrophage infiltration plays an important role in alteration of oxygen homeostasis in Aqp11 -/- mice.