Membrane Permeability in a Large Macrocyclic Peptide Driven by a Saddle-Shaped Conformation.

The effort to modulate challenging protein targets has stimulated interest in ligands that are larger and more complex than typical small-molecule drugs. While combinatorial techniques such as mRNA display routinely produce high-affinity macrocyclic peptides against classically undruggable targets, poor membrane permeability has limited their use toward primarily extracellular targets. Understanding the passive membrane permeability of macrocyclic peptides would, in principle, improve our ability to design libraries whose leads can be more readily optimized against intracellular targets. Here, we investigate the permeabilities of over 200 macrocyclic 10-mers using the thioether cyclization motif commonly found in mRNA display macrocycle libraries. We identified the optimal lipophilicity range for achieving permeability in thioether-cyclized 10-mer cyclic peptide-peptoid hybrid scaffolds and showed that permeability could be maintained upon extensive permutation in the backbone. In one case, changing a single amino acid from d-Pro to d-NMe-Ala, representing the loss of a single methylene group in the side chain, resulted in a highly permeable scaffold in which the low-dielectric conformation shifted from the canonical cross-beta geometry of the parent compounds into a novel saddle-shaped fold in which all four backbone NH groups were sequestered from the solvent. This work provides an example by which pre-existing physicochemical knowledge of a scaffold can benefit the design of macrocyclic peptide mRNA display libraries, pointing toward an approach for biasing libraries toward permeability by design. Moreover, the compounds described herein are a further demonstration that geometrically diverse, highly permeable scaffolds exist well beyond conventional drug-like chemical space.

Optimization of α-amido boronic acids via cryo-electron microscopy analysis: Discovery of a novel highly selective immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual inhibitor.

The immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit ß5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over ß5c may be desirable for treating autoimmune diseases. In this study, we present the optimization and discovery of α-amido boronic acids using cryo-electron microscopy (cryo-EM). The exploitation of structural differences between the proteasome subunits led to the identification of a highly selective LMP7/LMP2 dual inhibitor 19. Molecular dynamics simulation based on cryo-EM structures of the proteasome subunits complexed with 19 explained the inhibitory activity profile. In mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin, results indicate that 19 is orally bioavailable and shows promise as potential treatment for autoimmune diseases.

Potential mechanisms of tumor progression associated with postoperative infectious complications.

There is increasing evidence that postoperative infectious complications (PICs) are associated with poor prognosis after potentially curative surgery. However, the role that PICs play in tumor development remains unclear. In this article, we reviewed the literature for novel insights on the mechanisms of cancer progression associated with PICs. The Medline and EMBASE databases were searched for publications regarding the role of suppression of antitumor immunity by PIC in tumor progression and selected 916 manuscripts were selected for this review. In addition, a summary of the authors' own experimental data from this field was set in the context of current knowledge regarding cancer progression under septic conditions. Initially, sepsis/microbial infection dramatically activates the systemic immune system with increases in pro-inflammatory mediators, which results in the development of systemic inflammatory response syndrome; however, when sepsis persists in septic patients, a shift toward an anti-inflammatory immunosuppressive state, characterized by macrophage deactivation, reduced antigen presentation, T cell anergy, and a shift in the T helper cell pattern to a predominantly TH2-type response, occurs. Thus, various cytokine reactions and the immune status dynamically change during microbial infection, including PIC. We proposed three possible mechanisms for the tumor progression associated with PIC: first, a mechanism in which microbes and/or microbial PAMPs may be directly involved in cancer growth; second, a mechanism in which factors released from immunocompetent cells during infections may affect tumor progression; and third, a mechanism in which factors suppress host tumor immunity during infections, which may result in tumor progression. A more detailed understanding by surgeons of the immunological features in cancer patients with PIC can subsequently open new avenues for improving unfavorable long-term oncological outcomes associated with PICs.

Long-term outcomes of nasogastric tube with Gastrografin for adhesive small bowel obstruction.

BACKGROUND: We had previously reported that the administration of Gastrografin through a nasogastric tube (NGT-G) followed by long tube (LT) strategy could be a novel standard treatment for adhesive small bowel obstruction (ASBO); however, the long-term outcomes after initial improvement remain unknown. This study aimed to analyze the long-term outcomes of first-line NGT-G. METHODS: Enrolled patients with ASBO were randomly assigned to receive LT or NGT-G between July 2016 and November 2018. Thereafter, the cumulative surgery rate, cumulative recurrence rate, and overall survival (OS) rate were analyzed. In addition, subset analysis was conducted to determine the cumulative recurrence rate according to colonic contrast with Gastrografin at 24 h. RESULTS: A total of 223 patients (LT group, n = 111; NGT-G group, n = 112) were analyzed over a median follow-up duration of 550 days. The cumulative 1-year surgery rates, cumulative 1-year recurrence rates, and 1-year OS rates in the LT and NGT-G groups were 18.8% and 18.1%, 30.0% and 31.7%, and 99.1% and 96.6%, respectively; no significant differences were observed between both groups. In the NGT-G group, a negative colonic contrast at 24 h demonstrated a higher tendency for future recurrence compared with a positive colonic contrast at 24 h (1-year recurrence rate: negative contrast, 46.9% vs positive contrast, 27.6%). CONCLUSIONS: Gastrografin through a nasogastric tube followed by LT can be a promising treatment strategy for ASBO, with long-term efficacies equivalent to initial LT placement.

Survey on the current status of the indication and implementation protocols for bile replacement in patients with external biliary drainage with special reference to infection control.

PURPOSE: This survey of bile replacement (BR) was conducted on patients with external biliary drainage to assess the current status of indication and implementation protocol of BR with special reference to infection control. METHODS: A 12-item questionnaire regarding the performance of perioperative BR was sent to 124 institutions in Japan. RESULTS: BR was performed in 29 institutions, and the indication protocol was introduced in 19. BR was performed preoperatively in 11 institutions, pre- and postoperatively in 12, and postoperatively in 6. The methods used for BR administration included oral intake (n = 10), nasogastric tube (n = 1), enteral nutrition tube (n = 3), oral intake and enteral nutrition tube (n = 6), oral intake or nasogastric tube (n = 2), nasogastric tube and enteral nutrition tube (n = 2), and oral intake or nasogastric tube and enteral nutrition tube (n = 5). In 10 of 29 institutions, isolation of multidrug-resistant organisms and a high bacterial load were considered contraindications for the use of BR. Seven institutions experienced environmental contamination. CONCLUSIONS: Given the different implementation of BR among institutions, the appropriate indication and protocols for BR should be established for infection control.

Cyclosporin A: Conformational Complexity and Chameleonicity.

The chameleonic behavior of cyclosporin A (CsA) was investigated through conformational ensembles employing multicanonical molecular dynamics simulations that could sample the cis and trans isomers of N-methylated amino acids; these assessments were conducted in explicit water, dimethyl sulfoxide, acetonitrile, methanol, chloroform, cyclohexane (CHX), and n-hexane (HEX) using AMBER ff03, AMBER10:EHT, AMBER12:EHT, and AMBER14:EHT force fields. The conformational details were discussed employing the free-energy landscapes (FELs) at T = 300 K; it was observed that the experimentally determined structures of CsA were only a part of the conformational space. Comparing the ROESY measurements in CHX-d12 and HEX-d14, the major conformations in those apolar solvents were essentially the same as that in CDCl3 except for the observation of some sidechain rotamers. The effects of the metal ions on the conformations, including the cis/trans isomerization, were also investigated. Based on the analysis of FELs, it was concluded that the AMBER ff03 force field best described the experimentally derived conformations, indicating that CsA intrinsically formed membrane-permeable conformations and that the metal ions might be the key to the cis/trans isomerization of N-methylated amino acids before binding a partner protein.

[A Case of Advanced Gastric Cancer with Para-Aortic Lymph Node Metastasis with Long-Term Survival by Chemotherapy and Surgery].

Chemotherapy is the standard treatment for unresectable gastric cancer, but there is no clear evidence of therapeutic lymphadenectomy in conversion surgery after the tumor shrinks or the combined effect of perioperative chemotherapy. A 63-year-old man was diagnosed with advanced gastric cancer by upper gastrointestinal endoscopy; computed tomography (CT)showed swelling of the gastric regional lymph nodes, abdominal para-aortic lymph nodes, and left supraclavicular lymph node. After 4 courses of combination therapy with S-1 and cisplatin(SP therapy), CT showed that the left supraclavicular lymph node disappeared and the para-aortic lymph node was reduced. Distal gastrectomy and D2 plus para-aortic lymph node dissection were performed as conversion surgery. Two courses of postoperative SP therapy were administered, and S-1 monotherapy was continued for 2 years and 6 months. After 5 years and 1 month since the operation, the patient is alive without recurrence. This case shows that SP therapy can be effective as chemotherapy for unresectable gastric cancer. In addition, that conversion surgery after chemotherapy may contribute to recurrence-free survival.

High-Precision Atomic Charge Prediction for Protein Systems Using Fragment Molecular Orbital Calculation and Machine Learning.

Here, we have constructed neural network-based models that predict atomic partial charges with high accuracy at low computational cost. The models were trained using high-quality data acquired from quantum mechanics calculations using the fragment molecular orbital method. We have succeeded in obtaining highly accurate atomic partial charges for three representative molecular systems of proteins, including one large biomolecule (approx. 2000 atoms). The novelty of our approach is the ability to take into account the electronic polarization in the system, which is a system-dependent phenomenon, being important in the field of drug design. Our high-precision models are useful for the prediction of atomic partial charges and expected to be widely applicable in structure-based drug designs such as structural optimization, high-speed and high-precision docking, and molecular dynamics calculations.

Impact of perioperative high mobility group box chromosomal protein 1 expression on long-term outcomes in patients with esophageal squamous cell carcinoma.

BACKGROUND AND AIM: High mobility group box chromosomal protein-1 (HMGB-1) is a potential late mediator of sepsis and a possible risk factor for postoperative pulmonary complications after esophagectomy. This study aimed to determine the relationship between HMGB-1 and clinicopathological factors and long-term prognosis after esophagectomy for esophageal cancer. METHODS: We measured perioperative serum HMGB-1 levels using ELISA and HMGB-1 protein by immunohistochemistry expression in resected specimens. RESULTS: Postoperative serum HMGB-1 levels were significantly higher than preoperative levels. Preoperative serum HMGB-1 levels were significantly higher in patients with more intraoperative bleeding, longer intensive care unit stays, and postoperative pneumonia. Postoperative serum HMGB-1 levels were significantly higher in older patients and those with longer operation time and more intraoperative bleeding. There were significant differences in long-term outcomes according to postoperative but not preoperative serum HMGB-1 levels. Multivariate analysis demonstrated that advanced pathological stage, postoperative pulmonary complications, and higher postoperative serum HMGB-1 levels were independently associated with relapse-free survival and overall survival. Preoperative serum HMGB-1 levels were significantly higher in patients with high HMGB-1 expression than those with low HMGB-1 expression by immunohistochemistry, whereas such statistical differences were not observed in postoperative serum HMGB-1. There were no differences in relapse-free survival and overall survival according to HMGB-1 expression by immunohistochemistry. Serum HMGB-1 levels were significantly increased after esophagectomy for esophageal cancer. CONCLUSION: Elevated postoperative serum HMGB-1, which was associated not only with poor long-term but also short-term outcomes such as postoperative complications, might serve as a potential marker for prognosis in esophageal cancer.

Two Occurrences of Leukemia Relapse Due to Mismatched HLA Loss After Haploidentical Stem Cell Transplantation From Different Family Donors With KIR Ligand Mismatch.

Mismatched HLA loss is a cause of leukemia relapse after HLA-haploidentical stem cell transplantation (haplo-SCT). We report a patient with a history of 2 occurrences of leukemia relapse due to mismatched HLA loss after haplo-SCT. He received haplo-SCT from his father but showed leukemia relapse with loss of the maternal HLA haplotype. He then underwent haplo-SCT from his mother, and developed relapse with loss of the paternal HLA haplotype. Both donors had killer cell immunoglobulin-like receptor-ligand mismatch but alloreactive natural killer cells could not prevent relapse. Second haplo-SCT should be conducted carefully for patients with relapse due to mismatched HLA loss.

Drug-Like Properties in Macrocycles above MW 1000: Backbone Rigidity versus Side-Chain Lipophilicity.

Large macrocyclic peptides can achieve surprisingly high membrane permeability, although the properties that govern permeability in this chemical space are only beginning to come into focus. We generated two libraries of cyclic decapeptides with stable cross-ß conformations, and found that peptoid substitutions within the ß-turns of the macrocycle preserved the rigidity of the parent scaffold, whereas peptoid substitutions in the opposing ß-strands led to "chameleonic" species that were rigid in nonpolar media but highly flexible in water. Both rigid and chameleonic compounds showed high permeability over a wide lipophilicity range, with peak permeabilities differing significantly depending on scaffold rigidity. Our findings indicate that modulating lipophilicity can be used to engineer favorable ADME properties into both rigid and flexible macrocyclic peptides, and that scaffold rigidity can be used to tune optimal lipophilicity.

Endoscopic tissue shielding to prevent bleeding after endoscopic submucosal dissection: a prospective multicenter randomized controlled trial.

BACKGROUND: Bleeding after endoscopic submucosal dissection (ESD) is a severe adverse event. Recent reports have described the efficacy of the endoscopic shielding method with polyglycolic acid (PGA) sheets and fibrin glue for the prevention of adverse events after ESD. The aim of the present study was to investigate whether the PGA shielding method provides additional benefit in preventing post-ESD bleeding compared with standard care. METHODS: This was a prospective, multicenter, randomized controlled trial. Patients at high risk of post-ESD bleeding were enrolled in the study. Before ESD, patients were randomized to either the PGA group or the control group. After completing ESD in the PGA group, PGA sheets were placed onto the ulcer floor and adhered with fibrin glue. The primary end point was the post-ESD bleeding rate. RESULTS: 140 eligible patients were enrolled from September 2014 to September 2016, and 137 were included in the intention-to-treat analysis (67 in the PGA group and 70 in the control group). Post-ESD bleeding occurred in three patients (4.5 %) in the PGA group and in four patients (5.7 %) in the control group; there was no significant difference between the two groups (P > 0.99). Post-ESD bleeding tended to occur later in the control group than in the PGA group (median 12.5 days [range 8 - 14] vs. 2 days [range 0 - 7], respectively). CONCLUSION: The PGA shielding method did not demonstrate a significant effect on the prevention of post-ESD bleeding.

Conformation and Permeability: Cyclic Hexapeptide Diastereomers.

Conformational ensembles of eight cyclic hexapeptide diastereomers in explicit cyclohexane, chloroform, and water were analyzed by multicanonical molecular dynamics (McMD) simulations. Free-energy landscapes (FELs) for each compound and solvent were obtained from the molecular shapes and principal component analysis at T = 300 K; detailed analysis of the conformational ensembles and flexibility of the FELs revealed that permeable compounds have different structural profiles even for a single stereoisomeric change. The average solvent-accessible surface area (SASA) in cyclohexane showed excellent correlation with the cell permeability, whereas this correlation was weaker in chloroform. The average SASA in water correlated with the aqueous solubility. The average polar surface area did not correlate with cell permeability in these solvents. A possible strategy for designing permeable cyclic peptides from FELs obtained from McMD simulations is proposed.

Transduced caudal-type homeobox (CDX) 2/CDX1 can induce growth inhibition on CDX-deficient gastric cancer by rapid intestinal differentiation.

Intestinal metaplasia induced by ectopic expression of caudal-type homeobox (CDX)2 and/or CDX1 (CDX) is frequently observed around gastric cancer (GC). Abnormal expression of CDX is also observed in GC and suggests that inappropriate gastrointestinal differentiation plays essential roles in gastric tumorigenesis, but their roles on tumorigenesis remain unelucidated. Publicly available databases show that GC patients with higher CDX expression have significantly better clinical outcomes. We introduced CDX2 and CDX1 genes separately into GC-originated MKN7 and TMK1 cells deficient in CDX. Marked suppression of cell growth and dramatic morphological change into spindle-shaped flat form were observed along with induction of intestinal marker genes. G0-G1 growth arrest was accompanied by changed expression of cell cycle-related genes but not with apoptosis or senescence. Microarray analyses additionally showed decreased expression of gastric marker genes and increased expression of stemness-associated genes. Hierarchical clustering of 111 GC tissues and 21 non-cancerous gastric tissues by selected 18 signature genes based on our transcriptome analyses clearly categorized the 132 tissues into non-cancer, "CDX signature"-positive GC, and "CDX signature"-negative GC. Gene set enrichment analysis indicated that "CDX signature"-positive GC has lower malignant features. Immunohistochemistry of 89 GC specimens showed that 50.6% were CDX2-deficient, 66.3% were CDX1-deficient, and 44.9% were concomitant CDX2/CDX1-deficient, suggesting that potentially targetable GC cases by induced intestinal differentiation are quite common. In conclusion, exogenous expression of CDX2/CDX1 can lead to efficient growth inhibition of CDX-deficient GC cells. It is based on rapidly induced intestinal differentiation, which may be a future therapeutic strategy.

Impact of angiotensin-converting enzyme 2 levels on postoperative pneumonia after esophagectomy.

BACKGROUND: Pulmonary complications after esophagectomy are often fatal. The prediction of postoperative pulmonary complications remains a challenge. Accumulating evidence demonstrates a physiological and pathological role for angiotensin-converting enzyme 2 (ACE2) in the respiratory system. The purpose of this study was to evaluate the predictive value of ACE2 levels for the development of postoperative pneumonia. METHODS: To evaluate the association between serum ACE2 levels and pneumonia after esophagectomy, we retrospectively reviewed the medical records of 80 patients who underwent thoracoscopic esophagectomy for esophageal cancer from 2009 to 2014. RESULTS: Nineteen patients (23.8%) developed pneumonia after esophagectomy. Patients with pneumonia had significantly higher levels of ACE2 from the preoperative day to postoperative day (POD) 3, white blood cell count (POD7), and C-reactive protein (POD3, POD5, and POD7) than patients without pneumonia. Patients with postoperative pneumonia had higher serum ACE2 levels on POD3 than patients without pneumonia. CONCLUSIONS: The elevation of ACE2 levels on POD3 may predict the incidence of pneumonia.

Gastroesophageal Reflux Disease-Related Disorders of Systemic Sclerosis Based on the Analysis of 66 Patients.

BACKGROUND/AIMS: Gastroesophageal reflux disease (GERD)-related disorders of systemic sclerosis (SSc) patients have not been adequately investigated. METHODS: Sixty-six SSc patients (5 males and 61 females; 56.6 ± 14.6 years old) who underwent esophagogastroduodenoscopy were analyzed on the basis of 16 background factors. They were additionally compared with 116 matched non-SSc subjects controlling age, sex, and use of proton pump inhibitors (PPIs). RESULTS: The mean disease duration of 66 patients was 5.1 ± 8.1 years, and their breakdown was as follows: 53 (80.3%) with GERD, 38 (57.6%) with GERD-related symptoms, and 20 (30.3%) with reflux esophagitis (RE; LA-A: 10, LA-B: 5, LA-C: 4, LA-D: 1). Use of PPI (p = 0.0455), complication of interstitial lung disease (p = 0.0242), and history of cyclophosphamide therapy (p = 0.0184) denoted significant association with GERD-related symptoms. Older age (p = 0.0211) was significantly associated with RE. None of GERD-related disorders showed any difference between 37 diffuse cutaneous SSc and 29 limited cutaneous SSc patients. The matched analysis indicated that SSc patients had higher prevalence of GERD (p < 0.0001), GERD-related symptoms (p = 0.0034), and RE (p = 0.0002). CONCLUSION: SSc patients tend to have worse GERD symptoms and severer RE. However, most SSc-associated factors did not show significant association with GERD-related disorders, indicating the difficulty in predicting GERD-related disorders among SSc patients.

Expression of Gastric Markers Is Associated with Malignant Potential of Nonampullary Duodenal Adenocarcinoma.

BACKGROUND: Sporadic nonampullary duodenal epithelial tumors (NADETs) are uncommon, and thus their clinicopathological features have not been fully assessed. AIMS: In this study, we have analyzed a series of early sporadic NADETs, focusing on various immunohistological features. METHODS: We conducted a multicenter retrospective analysis of 68 patients with endoscopically resected sporadic NADETs. Associations between immunohistological features and clinicopathological features were statistically analyzed. RESULTS: The 68 patients consisted of 46 men (68%) and 22 women (32%) with a mean age of 60.7 ± 12.2 years (range 37-85 years). The 68 tumors were composed of 39 adenomas (57%) and 29 early-stage adenocarcinomas (43%). Duodenal adenocarcinomas were larger in size than adenomas and had papillary architecture in their pathological diagnosis with statistical significance. Duodenal adenocarcinomas also demonstrated a significantly higher expression of gastric markers (MUC5AC and MUC6) and a higher MIB-1 index. Duodenal adenomas were contrastively apt to express intestinal markers (MUC2, CDX1 and CDX2). Of the 68 cases analyzed, there were only 3 tumors positive for p53 staining, all of which were adenocarcinoma. When 7 submucosal invasive cancers and 21 intramucosal cancers were compared, submucosal invasion was positively associated with expression of MUC5AC. Also, submucosal invasion showed strong association with double-positivity of MUC5AC and MUC6. CONCLUSIONS: Our results indicate that immunohistochemical evaluation is useful for predicting malignant potential of NADETs, especially focusing on the expression of gastrointestinal markers.

Transparency-enhancing technology allows three-dimensional assessment of gastrointestinal mucosa: A porcine model.

Although high-resolution three-dimensional imaging of endoscopically resected gastrointestinal specimens can help elucidating morphological features of gastrointestinal mucosa or tumor, there are no established methods to achieve this without breaking specimens apart. We evaluated the utility of transparency-enhancing technology for three-dimensional assessment of gastrointestinal mucosa in porcine models. Esophagus, stomach, and colon mucosa samples obtained from a sacrificed swine were formalin-fixed and paraffin-embedded, and subsequently deparaffinized for analysis. The samples were fluorescently stained, optically cleared using transparency-enhancing technology: ilLUmination of Cleared organs to IDentify target molecules method (LUCID), and visualized using laser scanning microscopy. After observation, all specimens were paraffin-embedded again and evaluated by conventional histopathological assessment to measure the impact of transparency-enhancing procedures. As a result, microscopic observation revealed horizontal section views of mucosa at deeper levels and enabled the three-dimensional image reconstruction of glandular and vascular structures. Besides, paraffin-embedded specimens after transparency-enhancing procedures were all assessed appropriately by conventional histopathological staining. These results suggest that transparency-enhancing technology may be feasible for clinical application and enable the three-dimensional structural analysis of endoscopic resected specimen non-destructively. Although there remain many limitations or problems to be solved, this promising technology might represent a novel histopathological method for evaluating gastrointestinal cancers.

Abdominal Infection Suppresses the Number and Activity of Intrahepatic Natural Killer Cells and Promotes Tumor Growth in a Murine Liver Metastasis Model.

BACKGROUND: Increasing evidence suggests that postoperative infection is associated with poorer long-term outcome in various malignancies. However, the mechanism of poor prognosis induced by postoperative infection has not been clearly explained. We sought to determine whether abdominal infection promotes cancer metastases in a murine liver metastasis model, and to investigate the role of liver natural killer (NK) cells on antitumor immunity during abdominal infection. METHODS: Female BALB/c (8-10 weeks old) mice were inoculated with NL-17 colon cancer cells into the spleen and then subjected to abdominal infection induced by cecal ligation and puncture (CLP) or sham treatment. The extent of liver metastases and cytokine production in the serum and liver were investigated. Cell fraction and cytotoxic activities of liver mononuclear cells (MNCs) were elucidated. RESULTS: CLP mice had poorer survival and their serum levels of IL-6, -10, and -12p70 were significantly elevated on day 1 compared with sham-treated and control mice. No obvious differences in cytokine levels of the liver homogenates were identified among the three groups, except IL-12p70 levels in CLP mice on day 7 significantly decreased. The cytotoxic activities of liver MNCs were significantly suppressed in CLP mice soon after tumor inoculation. Flow cytometry revealed a decrease in NK cells in the liver and perforin and granzyme B expression levels. CONCLUSIONS: Abdominal infection promoted liver metastases in a murine liver metastasis model, which may be partially caused by a decrease in the number and activity of NK cells during abdominal infection.