Bowman's topography for improved detection of early ectasia

The aim of this study was to evaluate whether OCT topography of the Bowman's layer and artificial intelligence (AI) can result in better diagnosis of forme fruste (FFKC) and clinical keratoconus (KC). Normal (n = 221), FFKC (n = 72) and KC (n = 116) corneas were included. Some of the FFKC and KC patients had the fellow eye (VAE‐NT) with normal topography (n = 30). The Scheimpflug and OCT scans of the cornea were analyzed. The curvature and surface aberrations (ray tracing) of the anterior corneal surface [air‐epithelium (A‐E) interface in OCT] and epithelium‐Bowman's layer (E‐B) interface (in OCT only) were calculated. Four random forest models were constructed: (1) Scheimpflug only; (2) OCT A‐E only; (3) OCT E‐B only; (4) OCT A‐E and E‐B combined. For normal eyes, both Scheimpflug and OCT (A‐E and E‐B combined) performed equally in identifying these eyes (P = .23). However, OCT A‐E and E‐B showed that most VAE‐NT eyes were topographically similar to normal eyes and did not warrant a separate classification based on topography alone. For identifying FFKC eyes, OCT A‐E and E‐B combined performed significantly better than Scheimpflug (P = .006). For KC eyes, both Scheimpflug and OCT performed equally (P = 1.0). Thus, OCT Topography of Bowman's layer significantly improved the detection of FFKC eyes.     

Application of high resolution OCT to evaluate irregularity of Bowman's layer in asymmetric keratoconus

Irregularity of the Bowman's layer (BL) in keratoconus (KC, sample size (n) = 28) and forme fruste keratoconus (FFKC, n = 18) eyes was evaluated. Subjects underwent high resolution OCT imaging (Bioptigen Inc., USA) and corneal tomography (Pentacam v1.20r41, OCULUS Optikgeräte GmbH, Germany). Anterior edge of the BL was segmented. A Bowman's roughness index (BRI) was defined as the sum of the enclosed areas between segmented edge and a smooth 3^rd order polynomial fit to the edge. BRI was compared with corneal aberrations, Keratoconus percentage index (KISA), Cone location magnitude index (CLMI) and Belin–Ambrosio enhanced ectasia display overall deviation index (BAD‐D). Area under the curve (AUC) was determined with logistic regression (LR). Mean BRI (×10^–3) was 2.12 mm², 1.81 mm² and 1.7 mm² in normal (n = 26), FFKC and KC eyes, respectively (p < 0.001). BAD‐D (0.79) and BRI (0.74) had the best AUC for FFKC. By combining BAD‐D and BRI, the AUC improved to 0.85 (p = 0.01). For KC eyes, KISA (0.94), CLMI (0.88), BAD‐D (0.96) and aberrations had comparable AUC (p > 0.05). However, LR with BRI and other indices didn't improve the AUC in KC eyes (p > 0.05). BRI was significantly reduced in FFKC and KC eyes. It improved the detection of FFKC but not KC eyes.

     

Vascular changes precede tomographic changes in diabetic eyes without retinopathy and improve artificial intelligence diagnostics

The purpose of this study was to evaluate early vascular and tomographic changes in the retina of diabetic patients using artificial intelligence (AI). The study included 74 age‐matched normal eyes, 171 diabetic eyes without retinopathy (DWR) eyes and 69 mild non‐proliferative diabetic retinopathy (NPDR) eyes. All patients underwent optical coherence tomography angiography (OCTA) imaging. Tomographic features (thickness and volume) were derived from the OCTA B‐scans. These features were used in AI models. Both OCT and OCTA features showed significant differences between the groups (P < .05). However, the OCTA features indicated early retinal changes in DWR eyes better than OCT (P < .05). In the AI model using both OCT and OCTA features simultaneously, the best area under the curve of 0.91 ± 0.02 was obtained (P < .05). Thus, the combined use of AI, OCT and OCTA significantly improved the early diagnosis of diabetic changes in the retina.     

Corneal tomographic features of postrefractive surgery ectasia

The purpose of this study was to evaluate the tomographic features of postrefractive surgery eyes. This was a retrospective evaluation of clinical data. Three patients with post‐LASIK (laser‐assisted in situ keratomileusis) and two patients with post‐SMILE (small incision lenticule extraction) ectasia were imaged with Scheimpflug imaging (SI, Pentacam) and optical coherence tomography (OCT, RTVue). Curvature and wavefront aberrations of the air‐epithelium interface (A‐E) and epithelium‐Bowman's layer interface (E‐B) were derived. OCT of normal and keratoconic eyes from an earlier study were compared with the data of the ectasia eyes. Curvature and aberrometry of the A‐E interfaces were statistically similar between SI and OCT. However, OCT revealed a steeper and more aberrated E‐B interface than A‐E though correlation between them was inferior to the correlation for keratoconic eyes. Furthermore, the magnitude of differences between the A‐E and E‐B interfaces was greater in the ectasia eyes than the keratoconic eyes. OCT could possibly assist better in selecting appropriate treatment plan for postrefractive surgery ectasia eyes than conventional tomographers.      

Evaluation of corneal structures in myopic eyes more than twenty‐two years after photorefractive keratectomy

The aim of this study is to evaluate corneal epithelial thickness (CET), corneal densitometry (CD) in 84 myopic eyes (57 patients) more than 22 years after photorefractive keratectomy, using anterior segment‐optical coherence tomography (AS‐OCT) and Scheimpflug imaging system. The CET was significantly higher in all operated eyes than in unoperated eyes in central sector. A statistically significant increase in CD in corneal anterior layer of central sector was shown in groups of operated eyes with greater ablation depth respect to unoperated eyes. While there was no significant difference in CD between the operated eyes groups with lower ablation depth and unoperated eyes. A significant trend toward higher values in anterior CD with deeper ablations in central sector was found. These noninvasive imaging techniques allow to better understand the corneal remodeling process after photoablation and to monitor the patients over time.     

Quantification of diabetic macular ischemia using novel three‐dimensional optical coherence tomography angiography metrics

We applied three‐dimensional (3D) analysis to optical coherence tomography angiography (OCTA) to measure macular ischemia in eyes affected by non‐proliferative diabetic retinopathy (DR). A previously validated algorithm was applied to OCTA data in order to obtain 3D visualization of the retinal vasculature. Successively, a global thresholding algorithm was applied and two novel quantitative metrics were introduced: 3D vascular volume and 3D perfusion density. Two‐dimensional (2D) OCTA metrics were also obtained with different binarization thresholds for comparison. Of the 30 patients included, 15 were diagnosed with DR and 15 were controls. The 3D vascular volume and 3D perfusion density were reduced in DR eyes (P < .0001). The 2D variables also significantly differ between groups. The 3D perfusion density had the highest area under the receiver operating characteristic curve (0.964) among tested variables. Assessing quantitative perfusion using 3D analysis is reliable and promising, and with an elevated diagnostic efficacy in identifying DR eyes.     

Comparison of the Iowa Reference Algorithm to the Heidelberg Spectralis optical coherence tomography segmentation algorithm

For spectral‐domain optical coherence tomography (SD‐OCT) studies of neurodegeneration, it is important to understand how segmentation algorithms differ in retinal layer thickness measurements, segmentation error locations and the impact of manual correction. Using macular SD‐OCT images of frontotemporal degeneration patients and controls, we compare the individual and aggregate retinal layer thickness measurements provided by two commonly used algorithms, the Iowa Reference Algorithm and Heidelberg Spectralis, with manual correction of significant segmentation errors. We demonstrate small differences of most retinal layer thickness measurements between these algorithms. Outer sectors of the Early Treatment Diabetic Retinopathy Study grid require a greater percent of eyes to be corrected than inner sectors of the retinal nerve fiber layer (RNFL). Manual corrections affect thickness measurements mildly, resulting in at most a 5% change in RNFL thickness. Our findings can inform researchers how to best use different segmentation algorithms when comparing retinal layer thicknesses.     

Efficacy of cyhalothrin and lambdacyhalothrin against Boophilus microplus (Acari: Ixodidae).

A study was conducted to determine the efficacy of cyhalothrin and lambdacyhalothrin as a whole-body spray and lambdacyhalothrin as a pour-on application on cattle infested with Boophilus microplus (Canestrini). A laboratory bioassay was also done with both spray formulations. Cattle were infested with all parasitic life stages of B. microplus, then treated with 0.007 or 0.01% (AI) concentrations of cyhalothrin as a spray; 0.005, 0.007, or 0.01% (AI) concentrations of lambdacyhalothrin as a spray, or 1% (AI) lambdacyhalothrin as a pour-on. As determined by calculations of the index of reproduction (IR), the 0.007 and 0.01% (AI) cyhalothrin provided 92.4 and 97.3% control, respectively; the 0.005, 0.007, and 0.01% (AI) lambdacyhalothrin provided 92.4, 98.2 (average of two treatments) and 99.3% control, respectively; and the lambdacyhalothrin pour-on treatment provided < 50% control. Bioassay results indicated lambdacyhalothrin to be twice as effective as cyhalothrin against B. microplus.     

Increased migration of Langerhans cells in response to HPV16 E6 and E7 oncogene silencing: role of CCL20

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intraepithelial lesions (SILs) show quantitative and functional alterations of Langerhans cells (LC). The infiltration of immature LC in the squamous epithelium is mainly controlled by Macrophage Inflammatory Protein 3α/CCL20. After having shown that CCL20 production is altered in HPV-transformed keratinocytes (KC), the possible role of HPV16 E6 and E7 viral oncoproteins in the reduced CCL20 levels observed in SILs was investigated by silencing HPV16 E6 and E7 oncogenes by RNA interference (siRNA). This treatment not only increased CCL20 secretion but also resulted in the modulation of NF-κB p50, p52 and p65 precursor localization. Moreover, silencing of E6 and E7 oncogenes in HPV16-transformed KC induced a significantly higher migratory capacity of LC in a Boyden chamber assay and in an in vitro formed (pre)neoplastic epithelium reminiscent of high-grade SILs. Anti-CCL20 neutralizing antibody experiments showed that the increased migration of LC is due to the re-expression of CCL20 in E6 and E7 siRNA transfected KC. These data suggest that HPV16 E6/E7-induced down-regulation of CCL20 observed during the cervical carcinogenesis may contribute to a diminished capacity of the immune system to control HPV infection. P. Hubert and J. H. Caberg contributed equally to this work.     

Inside Cover: Optical coherence tomography as a noninvasive 3D real time imaging tool for the rapid evaluation of phenotypic variations in insect embryonic development (J. Biophotonics 2/2020)

Top: Illustration of how to obtain 3D embryo images from a locust egg using OCT B‐scans. The internal structures of the embryo can be clearly identified as it develops. Bottom: The real time observation of katatrepsis and twist of a lowland locust embryo on days 7‐8, as the embryo develops. The embryonic movements can be readily identified by tracking the positions of the embryo's eyes (E1 and E2). Further details can be found in the article by Ya Su, Liya Wei, Hao Tan, et al. ( e201960047 ).

     

An automated framework to quantify areas of regional ischemia in retinal vascular diseases with OCT angiography

In this observational and cross‐sectional study, capillary nonperfusion (CNP) and vascular changes in branch retinal vein occlusion (BRVO, sample size [n] = 26) and choroidal neovascularization (CNV, n = 29) were evaluated. Subjects underwent imaging using Optical coherence tomography angiography (Angiovue OCTA, RTVue XR, Optovue Inc., Fremont, California). Local fractal analysis was applied to the OCTA images of superficial, deep and choriocapillaris layer. CNP area (BRVO eyes) and vascular parameters were computed using local fractal‐based method. Sensitivity and specificity of vascular parameters were assessed with receiver operating characteristics curve. Automated CNP area showed excellent agreement with manually quantified CNP areas in both superficial (intraclass coefficient [ICC] = 0.96) and deep (ICC = 0.96) layers. BRVO eyes showed significantly altered (P < .05) vascular parameters in both superficial and deep layer as compared to normal eyes (n = 30). CNVM eyes had significantly higher capillary free zones (P < .001) as compared to normal eyes. In normal vs BRVO eyes, vessel density and spacing between the large vessels had similar area under the curve (AUC) (P > .05) in both superficial (0.97 and 0.97, respectively) and deep layer (0.99 and 0.98, respectively). Further, capillary free zones showed high AUC (0.92) in differentiating CNV eyes from normal eyes.      

Expression of SFRP Family Proteins in Human Keratoconus Corneas

We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (∼8 mm diameter) (n = 15) and whole control corneas (n = 7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC.     

Chlorpyrifos immersion to eliminate third instars of Japanese beetle (Coleoptera: Scarabaeidae) in balled and burlapped trees and subsequent treatment effects on red maple

This study examined chlorpyrifos immersion of balled and burlapped (B&B) nursery trees for elimination of third instars of Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), and for phytotoxicity on red maple, Acer rubrum L. Trees were harvested as 45- and 60-cm-diameter B&B and immersed in chlorpyrifos at U.S. Domestic Japanese Beetle Harmonization Plan rate (0.24 kg active ingredient [AI/100 liters) or lower rates of 0.015, 0.03, 0.06, and 0.12 kg (AI)/100 liters. The 0.03, 0.06, and 0.24 kg (AI) rates provided 100% control of Japanese beetle grubs in both 45- and 60-cm B&B. The 0.015 and 0.12 kg (AI) chlorpyrifos rates were 100% effective in three tests. However, in another test, 0.015 and 0.12 kg (AI) chlorpyrifos treatments had four (93% control) and one (98% control) grubs recovered, respectively. Root ball soils consisted of loam, silt loam, or clay loam texture classifications. Trunk diameter and internode growth of red maple harvested as 45-cm B&B decreased linearly with increasing chlorpyrifos dip rate during the first year, but effects were unapparent in the second year. Chlorpyrifos rates had no measurable impact on growth of red maples harvested as 60-cm B&B. No visual phytotoxicity symptoms were detected for chlorpyrifos rate or root ball size treatments. In conclusion, results support lowering the U.S. Domestic Japanese Beetle Harmonization Plan chlorpyrifos dip rate for category 2 states to at least 0.03 kg (AI) for B&B diameters < or =60 cm. Chlorpyrifos rates < 0.24 kg (AI) will lower cost, reduce worker exposure, and lessen potential environmental contamination.     

Comparison of oestrous synchronization regimens for lactating dairy cows.

The objective of this experiment was to evaluate various programmes for synchronization of oestrus. The focus of the study was to evaluate rates of detection of oestrus, synchrony of oestrus, pregnancy rate, and effect of ovarian status at initiation of the programmes on rates of detection of oestrus and pregnancy rate. Spring-calving, lactating dairy cows (n = 2009) were allocated at random to one of six treatments: (1) A (n = 335), progestogen (controlled intravaginal drug release; CIDR) inserted per vaginum 10 d before breeding season for 8 d, 10 microg of buserelin at CIDR insertion, PGF2alpha treatment on the day prior to CIDR removal, and AI of cows detected in oestrus within 6 d after CIDR withdrawal; (2) B (n = 330), as in A, plus 1 mg of oestradiol benzoate i.m. 10 h post CIDR withdrawal; (3) C (n = 347), as in A, except buserelin was replaced by 10 mg of oestradiol benzoate; (4) D (n = 335), as in A, plus PGF2alpha and oestradiol benzoate at CIDR insertion; (5) E (n = 332), CIDR containing a 10 mg oestradiol benzoate capsule inserted per vaginum for 12 d; or (6) F (n = 330), as in E, plus PGF2alpha on the day prior to CIDR withdrawal. The oestrous detection rate (number of cows detected in oestrus within 6 days of CIDR withdrawal as a proportion of the number of cows submitted for synchronization of oestrus) and oestrous synchrony (oestrous detection rate within 2 d of CIDR withdrawal), respectively, were greater (P<0.05) following B (95.7% of 330, 98.7% of 316) compared with any of the other programmes for synchronization of oestrus (A: 87.5 of 335, 79.4% of 293; C: 86.7% of 347, 80.0% of 301; D: 90.1% of 335, 89.8% of 302; E: 74.4% of 332, 70.4% of 247; F: 76.4% of 330, 78.5% of 252). The oestrous detection rate was reduced (P<0.05) among cows in metoestrus administered E (64.0% of 50) relative to similar cows administered F (82.8% of 64). Pregnancy rate was greater (P<0.05) following B (57.9% of 330) than A (48.9% of 335, P = 0.06), C (43.2% of 347), E (40.0% of 332), and F (35.1% of 330) but not D (59.3% of 302), when based on those cows presented for oestrous synchronization programmes. In conclusion, 1 mg of oestradiol benzoate administered 10 h post CIDR withdrawal (B) resulted in the best overall oestrous detection, oestrous synchrony, and pregnancy rates, which would be beneficial to a fixed-time AI program.     

Raman spectral discrimination in human liquid biopsies of oesophageal transformation to adenocarcinoma

The aim of this study was to determine whether Raman spectroscopy combined with chemometric analysis can be applied to interrogate biofluids (plasma, serum, saliva and urine) towards detecting oesophageal stages through to oesophageal adenocarcinoma [normal/squamous epithelium, inflammatory, Barrett's, low‐grade dysplasia, high‐grade dysplasia and oesophageal adenocarcinoma (OAC)]. The chemometric analysis of the spectral data was performed using principal component analysis, successive projections algorithm or genetic algorithm (GA) followed by quadratic discriminant analysis (QDA). The genetic algorithm quadratic discriminant analysis (GA‐QDA) model using a few selected wavenumbers for saliva and urine samples achieved 100% classification for all classes. For plasma and serum, the GA‐QDA model achieved excellent accuracy in all oesophageal stages (>90%). The main GA‐QDA features responsible for sample discrimination were: 1012 cm^?1 (C─O stretching of ribose), 1336 cm^?1 (Amide III and CH₂ wagging vibrations from glycine backbone), 1450 cm^?1 (methylene deformation) and 1660 cm^?1 (Amide I). The results of this study are promising and support the concept that Raman on biofluids may become a useful and objective diagnostic tool to identify oesophageal disease stages from squamous epithelium to OAC.     

Gender-influenced obesity QTLs identified in a cross involving the KK type II diabetes-prone mouse strain

The inheritance of adiposity and related traits has been investigated in the obese, diabetes-prone KK/HlLt (KK) and the lean, normoglycemic C57BL/6J (B6) mouse strains, their F₁ hybrids, and a large intercross generation. Adiposity index (AI) was defined as the sum of four fat depot weights divided by body weight. Both male and female KK mice were obese, but AI values averaged twofold higher in females than in males. In contrast, B6 females were slightly more lean than males. A genome-wide search revealed several qualitative trait loci (QTLs) affecting AI. The proximal region of Chromosome (Chr) 9 has a large effect on AI, with a much stronger effect in females (lod = 6.3) than in males (lod = 2.7). The data for females fit a model in which a dominant allele from KK increases AI by 30%, with the lod score peak falling between markers D9Mit66 and D9Mit328. This QTL has large effects on inguinal and mesenteric fat pad weights, with smaller effects on gonadal and retroperitoneal fat pads. The region of Chr 9 containing this QTL has extensive homology to human Chr 11q. An X-linked QTL affecting AI was evident in males (lod = 3.77), but not females (lod = 0.7). Exclusion of mesenteric fat from male AI resulted in an increased lod score (lod = 5.0) at 8 cM distal to DXMit166. A suggestive AI QTL (lod = 4.2), differentially affecting males, was localized to Chr 18 near the glucocorticoid receptor locus. A region of Chr 7 had a strong effect on body weight (lod = 6.9), a significant effect on inguinal fat% (lod = 4.4), and a suggestive effect on AI in females (lod = 4.1). Plasma leptin levels were associated with genotypes on Chr 9 (lod = 5.9) and Chr 7 (lod = 4.2). A region of Chr 1 had a suggestive effect on fasted blood glucose (lod = 3.6). Received: 23 March 1999 / Accepted: 2 June 1999     

Epithelium segmentation and automated Gleason grading of prostate cancer via deep learning in label‐free multiphoton microscopic images

In the current clinical care practice, Gleason grading system is one of the most powerful prognostic predictors for prostate cancer (PCa). The grading system is based on the architectural pattern of cancerous epithelium in histological images. However, the standard procedure of histological examination often involves complicated tissue fixation and staining, which are time‐consuming and may delay the diagnosis and surgery. In this study, label‐free multiphoton microscopy (MPM) was used to acquire subcellular‐resolution images of unstained prostate tissues. Then, a deep learning architecture (U‐net) was introduced for epithelium segmentation of prostate tissues in MPM images. The obtained segmentation results were then merged with the original MPM images to train a classification network (AlexNet) for automated Gleason grading. The developed method achieved an overall pixel accuracy of 92.3% with a mean F1 score of 0.839 for epithelium segmentation. By merging the segmentation results with the MPM images, the accuracy of Gleason grading was improved from 72.42% to 81.13% in hold‐out test set. Our results suggest that MPM in combination with deep learning holds the potential to be used as a fast and powerful clinical tool for PCa diagnosis.     

Developmental expression of the calcium‐activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium

Calcium‐activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium‐activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 657–675, 2014     

Optical coherence tomography image denoising using a generative adversarial network with speckle modulation

Optical coherence tomography (OCT) is widely used for biomedical imaging and clinical diagnosis. However, speckle noise is a key factor affecting OCT image quality. Here, we developed a custom generative adversarial network (GAN) to denoise OCT images. A speckle‐modulating OCT (SM‐OCT) was built to generate low speckle images to be used as the ground truth. In total, 210 000 SM‐OCT images were used for training and validating the neural network model, which we call SM‐GAN. The performance of the SM‐GAN method was further demonstrated using online benchmark retinal images, 3D OCT images acquired from human fingers and OCT videos of a beating fruit fly heart. The denoise performance of the SM‐GAN model was compared to traditional OCT denoising methods and other state‐of‐the‐art deep learning based denoise networks. We conclude that the SM‐GAN model presented here can effectively reduce speckle noise in OCT images and videos while maintaining spatial and temporal resolutions.