A Novel Pneumococcal Surface Protein K of Nonencapsulated Streptococcus pneumoniae Promotes Transmission among Littermates in an Infant Mouse Model with Influenza A Virus Coinfection.

We established an infant mouse model for colonization and transmission by nonencapsulated Streptococcus pneumoniae (NESp) strains to gain important information about its virulence among children. Invasive pneumococcal diseases have decreased dramatically since the worldwide introduction of pneumococcal capsular polysaccharide vaccines. Increasing prevalence of nonvaccine serotypes, including NESp, has been highlighted as a challenge in treatment strategy, but the virulence of NESp is not well understood. Protective strategies against NESp colonization and transmission between children require particularly urgent evaluation. NESp lacks capsules, a major virulence factor of pneumococci, but can cause a variety of infections in children and older people. PspK, a specific surface protein of NESp, is a key factor in establishing nasal colonization. In our infant mouse model for colonization and transmission by NESp strains, NESp could establish stable nasal colonization at the same level as encapsulated serotype 6A in infant mice and could be transmitted between littermates. Transmission was promoted by NESp surface virulence factor PspK and influenza virus coinfection. However, PspK deletion mutants lost the ability to colonize and transmit to new hosts. Promotion of NESp transmission by influenza was due to increased susceptibility of the new hosts. PspK was a key factor not only in establishment of nasal colonization but also in transmission to new hosts. PspK may be targeted as a new candidate vaccine for NESp infection in children.

Impact of the practical guideline on appropriate usage of antimicrobial treatments for surgical site infections in reconstructive surgery of head and neck cancer.

OBJECTIVES: In 2016, Japanese Society of Chemotherapy and Japan Society for Surgical Infection presented the practical guideline for appropriate usage of antimicrobial agents to prevent postoperative infections. This study aims to exhibit the validity of the guideline as a series of effective strategies for prevention of surgical site infections (SSIs) during reconstructive surgery of the head and neck cancer. METHODS: We retrospectively evaluated patients who underwent head and neck reconstructive surgery with free or pedicle flaps in a single institute in Japan between July 2010 and July 2020. We evaluated the incidence of SSIs, patient backgrounds, and microbiological characteristics on the basis of antimicrobial prophylaxis recommended by the guideline. RESULTS: Enrolled in this study were 102 patients in our institution who underwent head and neck reconstructive surgery with free or pedicle flaps between July 2010 and July 2020. In the period between January 2018 to July 2020 after the SSI guideline was advocated (SSI guideline period), the ratio of administration of sulbactam/ampicillin (SBT/ABPC) was significantly higher (P < 0.001) and the duration of prophylactic antimicrobial treatment was significantly shorter than in the period between July 2010 to December 2017 before the SSI guideline was advocated (Pre-SSI guideline period) (P < 0.001). Incidence of SSIs were similar, even when antibiotic use was changed to be short-term single-agent administration in accordance with the practical guideline. CONCLUSIONS: Adherence to the current Japanese practical guideline on appropriate antimicrobial prophylaxis for SSIs can shorten the duration of usage of antimicrobial treatment without increasing the risk for occurrence of SSIs.

Detecting the presence of supernumerary teeth during the early mixed dentition stage using deep learning algorithms: A pilot study.

BACKGROUND: Supernumerary teeth are a common anomaly and are frequently observed in paediatric patients. To prevent or minimize complications, early diagnosis and treatment is ideal in children with supernumerary teeth. AIM: This study aimed to apply convolutional neural network (CNN)-based deep learning to detect the presence of supernumerary teeth in children during the early mixed dentition stage. DESIGN: Three CNN models, AlexNet, VGG16-TL, and InceptionV3-TL, were employed in this study. A total of 220 panoramic radiographs (from children aged 6 years 0 months to 9 years 6 months) including supernumerary teeth (cases, n = 120) or no anomalies (controls, n = 100) were retrospectively analyzed. The CNN performances were assessed according to accuracy, sensitivity, specificity, receiver operating characteristic (ROC) curves, and area under the ROC curves for a cross-validation test dataset. RESULTS: The VGG16-TL model had the highest performance according to accuracy, sensitivity, specificity, and area under the ROC curve, but the other models had similar performance. CONCLUSION: CNN-based deep learning is a promising approach for detecting the presence of supernumerary teeth during the early mixed dentition stage.

Pneumococcal capsular phase shift is associated with invasion into cell-to-cell junctions and is inhibited by macrolides.

Streptococcus pneumoniae frequently colonizes the human nasopharynx beginning in the early childhood. Pneumococci exhibit a spontaneous and reversible phase shift between opaque and transparent allowing them to adapt to different environments. This is the first report of the dynamics of pneumococcal phase shift during the course of adhesion and subsequent invasion into epithelial cell monolayers by bacteria-cell co-culture assay with a time-lapse microscopy. The invasion of an inoculum between the human epithelial cells was dependent on the transparent phenotype, but successful replication of the cells within the cell layer was strongly associated with its transformation into an opaque-like variant. We also observed that sub-MIC levels of clarithromycin inhibited the spontaneous pneumococcal phase shift. Our results show that the pneumococcus can modulate its fitness in part because it can switch phenotype in response to the environment during not only inflammation but also during the establishment of colonization. Our current findings provide a more in depth understanding not only of how the pneumococcal phase shift acts to protect pneumococci from commensal flora and the immune status of the host, but also illustrate a novel strategy for antimicrobial treatments to interfere with pneumococcal colonization.

The phosphatidylethanolamine biosynthesis pathway provides a new target for cancer chemotherapy.

BACKGROUND & AIMS: Since human induced pluripotent stem cells (iPSCs) develop into hepatic organoids through stages that resemble human embryonic liver development, they can be used to study developmental processes and disease pathology. Therefore, we examined the early stages of hepatic organoid formation to identify key pathways affecting early liver development. METHODS: Single-cell RNA-sequencing and metabolomic analysis was performed on developing organoid cultures at the iPSC, hepatoblast (day 9) and mature organoid stage. The importance of the phosphatidylethanolamine biosynthesis pathway to early liver development was examined in developing organoid cultures using iPSC with a CRISPR-mediated gene knockout and an over the counter medication (meclizine) that inhibits the rate-limiting enzyme in this pathway. Meclizine's effect on the growth of a human hepatocarcinoma cell line in a xenotransplantation model and on the growth of acute myeloid leukemia cells in vitro was also examined. RESULTS: Transcriptomic and metabolomic analysis of organoid development indicated that the phosphatidylethanolamine biosynthesis pathway is essential for early liver development. Unexpectedly, early hepatoblasts were selectively sensitive to the cytotoxic effect of meclizine. We demonstrate that meclizine could be repurposed for use in a new synergistic combination therapy for primary liver cancer: a glycolysis inhibitor reprograms cancer cell metabolism to make it susceptible to the cytotoxic effect of meclizine. This combination inhibited the growth of a human liver carcinoma cell line in vitro and in a xenotransplantation model, without causing significant side effects. This drug combination was also highly active against acute myeloid leukemia cells. CONCLUSION: Our data indicate that phosphatidylethanolamine biosynthesis is a targetable pathway for cancer; meclizine may have clinical efficacy as a repurposed anti-cancer drug when used as part of a new combination therapy. LAY SUMMARY: The early stages of human liver development were modeled using human hepatic organoids. We identified a pathway that was essential for early liver development. Based upon this finding, a novel combination drug therapy was identified that could be used to treat primary liver cancer and possibly other types of cancer.

Humanized thymidine kinase-NOG mice can be used to identify drugs that cause animal-specific hepatotoxicity: a case study with furosemide.

Interspecies differences have limited the predictive utility of toxicology studies performed using animal species. A drug that could be a safe and effective treatment in humans could cause toxicity in animals, preventing it from being used in humans. We investigated whether the use of thymidine kinase (TK)-NOG mice with humanized livers could prevent this unfortunate outcome (i.e., "rescue" a drug for use in humans). A high dose of furosemide is known to cause severe liver toxicity in mice, but it is a safe and effective treatment in humans. We demonstrate that administration of a high dose of furosemide (200 mg/kg i.p.) causes extensive hepatotoxicity in control mice but not in humanized TK-NOG mice. This interspecies difference results from a higher rate of production of the toxicity-causing metabolite by mouse liver. Comparison of their survival curves indicated that the humanized mice were more resistant than control mice to the hepatotoxicity caused by high doses of furosemide. In this test case, humanized TK-NOG mouse studies indicate that humans could be safely treated with a high dose of furosemide.

Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity.

Due to the substantial interspecies differences in drug metabolism and disposition, drug-induced liver injury (DILI) in humans is often not predicted by studies performed in animal species. For example, a drug (bosentan) used to treat pulmonary artery hypertension caused unexpected cholestatic liver toxicity in humans, which was not predicted by preclinical toxicology studies in multiple animal species. In this study, we demonstrate that NOG mice expressing a thymidine kinase transgene (TK-NOG) with humanized livers have a humanized profile of biliary excretion of a test (cefmetazole) drug, which was shown by an in situ perfusion study to result from interspecies differences in the rate of biliary transport and in liver retention of this drug. We also found that readily detectable cholestatic liver injury develops in TK-NOG mice with humanized livers after 1 week of treatment with bosentan (160, 32, or 6 mg/kg per day by mouth), whereas liver toxicity did not develop in control mice after 1 month of treatment. The laboratory and histologic features of bosentan-induced liver toxicity in humanized mice mirrored that of human subjects. Because DILI has become a significant public health problem, drug safety could be improved if preclinical toxicology studies were performed using humanized TK-NOG.

Image preprocessing with contrast-limited adaptive histogram equalization improves the segmentation performance of deep learning for the articular disk of the temporomandibular joint on magnetic resonance images.

OBJECTIVES: The objective was to evaluate the robustness of deep learning (DL)-based encoder-decoder convolutional neural networks (ED-CNNs) for segmenting temporomandibular joint (TMJ) articular disks using data sets acquired from 2 different 3.0-T magnetic resonance imaging (MRI) scanners using original images and images subjected to contrast-limited adaptive histogram equalization (CLAHE). STUDY DESIGN: In total, 536 MR images from 49 individuals were examined. An expert orthodontist identified and manually segmented the disks in all images, which were then reviewed by another expert orthodontist and 2 expert oral and maxillofacial radiologists. These images were used to evaluate a DL-based semantic segmentation approach using an ED-CNN. Original and preprocessed CLAHE images were used to train and validate the models whose performances were compared. RESULTS: Original and CLAHE images acquired on 1 scanner had pixel values that were significantly darker and with lower contrast. The values of 3 metrics-the Dice similarity coefficient, sensitivity, and positive predictive value-were low when the original MR images were used for model training and validation. However, these metrics significantly improved when images were preprocessed with CLAHE. CONCLUSIONS: The robustness of the ED-CNN model trained on a dataset obtained from a single device is low but can be improved with CLAHE preprocessing. The proposed system provides promising results for a DL-based, fully automated segmentation method for TMJ articular disks on MRI.

Neuron Navigator 1 (Nav1) regulates the response to cocaine in mice.

Genetic variation accounts for much of the risk for developing a substance use disorder, but the underlying genetic factors and their genetic effector mechanisms are mostly unknown. Inbred mouse strains exhibit substantial and heritable differences in the extent of voluntary cocaine self-administration. Computational genetic analysis of cocaine self-administration data obtained from twenty-one inbred strains identified Nav1, a member of the neuron navigator family that regulates dendrite formation and axonal guidance, as a candidate gene. To test this genetic hypothesis, we generated and characterized Nav1 knockout mice. Consistent with the genetic prediction, Nav1 knockout mice exhibited increased voluntary cocaine intake and had increased motivation for cocaine consumption. Immunohistochemistry, electrophysiology, and transcriptomic studies were performed as a starting point for investigating the mechanism for the Nav1 knockout effect. Nav1 knockout mice had a reduced inhibitory synapse density in their cortex, increased excitatory synaptic transmission in their cortex and hippocampus, and increased excitatory neurons in a deep cortical layer. Collectively, our results indicate that Nav1 regulates the response to cocaine, and we identified Nav1 knockout induced changes in the excitatory and inhibitory synaptic balance in the cortex and hippocampus that could contribute to this effect.

The Role of Anatomical Imaging and Intraoperative Neuromonitoring (IONM) for Successful Prediction of a Nonrecurrent Laryngeal Nerve.

A nonrecurrent laryngeal nerve (NRLN) is a rare anatomical variant of laryngeal nerves that branches directly from the vagus nerve. The anatomical abnormality makes it difficult to identify the NRLN and results in high incidence of accidental nerve injury during surgery. A 76-year-old woman complained of swelling in the right side of her neck and visited our university hospital for further examination. Ultrasonography showed a right thyroid lobe mass with calcification and fine needle aspiration biopsy was classified as class III. Computed tomography revealed that the right subclavian artery branched directly from the descending aorta without branching from the brachiocephalic artery and ran behind the esophagus. Since it was afraid that the accidental injury of NRLN was likely to occur, a right thyroid lobe dissection using intraoperative neuromonitoring (IONM) was performed. After separating the connective tissue on the thyroid capsule from the right side of the trachea to the inferior pole laterally, the NRLN running across the level of the inferior margin of the cricoid cartilage was identified by using IONM 0.5 mA stimulation. After complete dissection of right thyroid lobe, we again stimulated the NRLN by 0.5 mA and the electromyographic response was confirmed. The pathological analysis confirmed nodular hyperplasia without malignancy; the condition was diagnosed as an adenomatous goiter. There was no vocal cord dysfunction and hoarseness after the surgery. IONM contributed to the prevention of NRLN injury during the surgery. We believe that it is important to confirm the presence or absence of an aberrant subclavian artery on preoperative imaging, and that IONM should be considered to identify the NRLN to prevent vocal cord paralysis if its presence is suspected.

Automated segmentation of articular disc of the temporomandibular joint on magnetic resonance images using deep learning.

Temporomandibular disorders are typically accompanied by a number of clinical manifestations that involve pain and dysfunction of the masticatory muscles and temporomandibular joint. The most important subgroup of articular abnormalities in patients with temporomandibular disorders includes patients with different forms of articular disc displacement and deformation. Here, we propose a fully automated articular disc detection and segmentation system to support the diagnosis of temporomandibular disorder on magnetic resonance imaging. This system uses deep learning-based semantic segmentation approaches. The study included a total of 217 magnetic resonance images from 10 patients with anterior displacement of the articular disc and 10 healthy control subjects with normal articular discs. These images were used to evaluate three deep learning-based semantic segmentation approaches: our proposed convolutional neural network encoder-decoder named 3DiscNet (Detection for Displaced articular DISC using convolutional neural NETwork), U-Net, and SegNet-Basic. Of the three algorithms, 3DiscNet and SegNet-Basic showed comparably good metrics (Dice coefficient, sensitivity, and positive predictive value). This study provides a proof-of-concept for a fully automated deep learning-based segmentation methodology for articular discs on magnetic resonance images, and obtained promising initial results, indicating that the method could potentially be used in clinical practice for the assessment of temporomandibular disorders.

A case of concurrent occurrence of carcinoma showing thymus-like differentiation and follicular variant of papillary thyroid cancer in the same thyroid.

Carcinoma showing thymus-like differentiation (CASTLE) is a rare thyroid cancer. This is the first report of a case of concurrent occurrence of CASTLE with papillary thyroid carcinoma (PTC). A 66-year-old male patient had hoarseness with right vocal cord paralysis. Ultrasonography revealed a hypoechoic nodule in the inferior pole of the right thyroid lobe. Ultrasound-guided fine-needle aspiration cytology suggested differentiated thyroid cancer. The patient underwent total thyroidectomy with neck dissection. Pathological examination revealed two different thyroid cancers: a CASTLE and a follicular variant of PTC. Postoperative radiation therapy was performed. The patient was still alive after 5 year following the initial treatment without evidence of recurrence. The oncological management of patients with concurrent occurrence of different thyroid cancers should consider the biological behavior of both tumors.

Lateral approach contributes to shortened surgical time in video-assisted neck surgery (VANS) for thyroid nodule.

Background: Despite endoscopic thyroid surgery contributing to excellent cosmetic outcomes, it requires longer surgical time than open surgery. This study evaluates the factors associated with operative time in video-assisted neck surgery. Methods: We retrospectively reviewed patients who underwent hemithyroidectomy by video-assisted neck surgery at a single hospital between 2017 and 2021. The clinical parameters were evaluated: age, sex, body weight, body mass index, coexistence of Hashimoto thyroiditis, side of operation, surgical approach to thyroid (midline approach or lateral approach), operation time, amount of blood loss during surgery, maximum tumor diameter and tumor volume, and postoperative complications. These parameters were compared to investigate the association of operative time and surgical approach. Results: Seventy consecutive patients were enrolled in this study, the median age at operation was 50 years, and there were 6 (8.6%) men and 64 (91.4%) women. The median body weight and body mass index at operation were 56.6 kg and 21.5, respectively. Coexistence of Hashimoto thyroiditis was found in 17 (24.3%) patients. Significant differences were found between the groups with longer and shorter operation time than median 201 minutes in surgical approaches to the thyroid (P < .001) and the amount of bleeding during surgery (P = .039). There were no differences in other candidates between the groups. Median operation times were 242 minutes in midline approach and 131 minutes in lateral approach (P < .001). Conclusion: The lateral approach of video-assisted neck surgery to the thyroid can contribute to shortened surgical time in the case of benign thyroid nodules.

Role of a tyrosine phosphorylation of SMG-9 in binding of SMG-9 to IQGAP and the NMD complex.

SMG-9 is a component of the NMD complex, a heterotetramer that also includes SMG-1 and SMG-8 in the complex. SMG-9 was also originally identified as a tyrosine-phosphorylated protein but the role of the phosphorylation is not yet known. In this study, we determined that IQGAP protein, an actin cytoskeleton modifier acts as a binding partner with SMG-9 and this binding is regulated by phosphorylation of SMG-9 at Tyr-41. SMG-9 is co-localized with IQGAP1 as a part of the process of actin enrichment in non-stimulated cells, but not in the EGF-stimulated cells. Furthermore, an increase in the ability of SMG-9 to bind to SMG-8 occurs in response to EGF stimulation. These results suggest that tyrosine phosphorylation of SMG-9 may play a role in the formation of the NMD complex in the cells stimulated by the growth factor.

Landmark annotation and mandibular lateral deviation analysis of posteroanterior cephalograms using a convolutional neural network.

BACKGROUND/PURPOSE: Facial asymmetry is relatively common in the general population. Here, we propose a fully automated annotation system that supports analysis of mandibular deviation and detection of facial asymmetry in posteroanterior (PA) cephalograms by means of a deep learning-based convolutional neural network (CNN) algorithm. MATERIALS AND METHODS: In this retrospective study, 400 PA cephalograms were collected from the medical records of patients aged 4 years 2 months-80 years 3 months (mean age, 17 years 10 months; 255 female patients and 145 male patients). A deep CNN with two optimizers and a random forest algorithm were trained using 320 PA cephalograms; in these images, four PA landmarks were independently identified and manually annotated by two orthodontists. RESULTS: The CNN algorithms had a high coefficient of determination (R 2 ), compared with the random forest algorithm (CNN-stochastic gradient descent, R 2  = 0.715; CNN-Adam, R 2  = 0.700; random forest, R 2  = 0.486). Analysis of the best and worst performances of the algorithms for each landmark demonstrated that the right latero-orbital landmark was most difficult to detect accurately by using the CNN. Based on the annotated landmarks, reference lines were defined using an algorithm coded in Python. The CNN and random forest algorithms exhibited similar accuracy for the distance between the menton and vertical reference line. CONCLUSION: Our findings imply that the proposed deep CNN algorithm for detection of facial asymmetry may enable prompt assessment and reduce the effort involved in orthodontic diagnosis.

A Rare Case of Thyroid Carcinoma Showing Thymus-Like Differentiation in a Young Adult.

Thyroid carcinoma showing thymus-like differentiation (CASTLE) is thought to originate from ectopic thymic tissue or remnants of the developing thymus within or adjacent to the thyroid. This case report describes a mass located on the left thyroid of a 28-year-old man. Fine-needle aspiration cytology revealed a number of lymphoid cells without atypia that were similar to those seen in a malignant lymphoma of the thyroid, and surgery was performed. Based on additional histopathological findings, the tumor was finally diagnosed as a CASTLE. It is difficult to diagnose this neoplasm using fine-needle aspiration cytology. However, it is possible to differentially diagnose CASTLE based on its histological features. CD5 is useful for diagnosing CASTLE with immunohistochemical staining.

Novel Antimicrobial Treatment Strategy Based on Drug Delivery Systems for Acute Otitis Media.

Despite tremendous success of pneumococcal conjugated vaccine and antimicrobial treatment by amoxicillin, acute otitis media (AOM) still remains as a great medical concern. Failure of antimicrobial treatment includes several factors. The middle ear cavity is surrounded by bone tissue, which makes it difficult to maintain sufficient concentration of antibiotics. Tympanic membrane of AOM patients thickens and actually becomes a barrier for topical therapy. This review discusses novel antimicrobial treatment strategies based on drug delivery systems (DDS) for AOM. To deliver drugs enough to kill the pathogenic bacteria without systemic side effects, the development of new antimicrobial treatment strategy applying innovative drug DDS has been expected. The sustained-release DDS can achieve sufficient time for antimicrobial concentrations to exceed minimum inhibitory concentration (MIC) for time-dependent antibiotics as well as enough maximum concentration for dose-dependent antibiotics to eradicate causative pathogens in the middle ear. The development of trans-tympanic membranes of DDS, such as hydrogels with chemical permeation enhancers (CPEs), is another attractive strategy. Phage is a promising strategy for developing DDS-based therapies. The DDS formulations enable antimicrobial treatment of AOM by a single dose and thus, an attractive future antimicrobial treatment for AOM.

Starplasty contributes to reduce tracheostomal granulation in pediatric tracheostomy.

BACKGROUND: Starplasty tracheostomy for pediatric patients has been suggested to reduce complications, including accidental decannulation and granulation. OBJECTIVES: This study, based in a single hospital, aims to evaluate whether starplasty tracheostomy decreases the incidence of postoperative granulation of tracheostoma. MATERIAL AND METHODS: A retrospective review was performed of patients that underwent tracheostomy under the age of 10 years in a single center between January 2001 and August 2020. RESULTS: Of the 46 patients reviewed, 18 were males and 28 were females, and the median age at the initial operation was 6 months. Methods of tracheostomy were starplasty in 16 patients, vertical in 15 patients, horizontal H-shaped in 10 patients, fenestration in 3 patients, and trap door/inverted U-shaped in two patients. During observation, tracheostoma granulation was found in 25 patients and bleeding from tracheostoma occurred in one patient. No other major complications were observed. The incidence of postoperative tracheostoma granulation was significantly lower in patients that underwent starplasty tracheostomy compared with patients that underwent other types of tracheostomy (p = .007). There was no difference in survival outcomes or ratio of decannulations. CONCLUSIONS: Starplasty tracheostomy was shown to decrease the incidence of tracheostoma granulation compared with other types of tracheostomy.

A rare case of the recurrent surgery for cribriform-morular variant of papillary thyroid carcinoma.

INTRODUCTION: The cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC) is an uncommon subtype of PTC, and it is associated with familial adenomatous polyposis (FAP). PRESENTATION OF CASE: We describe a 32-year-old female who presented recurrent CMV-PTC with FAP. Surgery performed after the recurrent disease resulted in left recurrent laryngeal nerve palsy due to formation of strong scar after initial operation. The histopathological examination revealed the recurrent CMV-PTC without thyroid capsular invasion in remnant thyroid tissue of berry ligament. In addition, the immunohistochemical analysis showed that ß-Catenin was diffusely positive in both cytoplasm and nucleus in CMV-PTC tissue. CONCLUSION: Total thyroidectomy in CMV-PTC with FAP should be performed at initial surgery due to high recurrence.

Rapamycin, but not cyclosporine or FK506, alters natural killer cell function.

Infiltration of natural killer (NK) cells into solid organ allografts is observed in clinical and experimental transplantation. Studies suggest a role for NK cells in acute and chronic rejection of solid organ allografts; however, the effects of immunosuppressive agents on NK cells are not clearly established. Rat NK cell lines were analyzed for proliferation and cytotoxicity in the presence of cyclosporine, FK506, or rapamycin. Lewis recipients of DA liver allografts received immunosuppressive agents after transplantation. NK cells demonstrated robust function both in the absence and presence of cyclosporine and FK506. In contrast, rapamycin significantly inhibited proliferation and cytotoxicity of NK cells. NK cell numbers remained stable in graft recipients treated with cyclosporine and FK506, whereas there was a significant decrease in NK cells in rapamycin-treated recipients. These data indicate that immunosuppressive drugs have differential effects on NK cell function that may impact the immune response of transplant recipients.