Design of complicated all-α protein structures.

A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. Here, to expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing 'difficult-to-describe' α-helical protein structures composed of irregularly aligned α-helices like globins. Backbone structure libraries consisting of a myriad of α-helical structures with five or six helices were generated by combining 18 helix-loop-helix motifs and canonical α-helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated α-helical proteins are created using typical building blocks. The method we developed will enable us to explore the universe of protein structures for designing novel functional proteins.

Safety and outcome of three-dimensional transoral videolaryngoscopic surgery.

BACKGROUND: Transoral videolaryngoscopic surgery (TOVS) is widely used in Japan, and conventional two-dimensional (2D) endoscopic methods have been established. Three-dimensional (3D) endoscopic surgery offers superior distance perception because it provides stereoscopic views. Recently, we have developed 3D endoscopy for TOVS (3D TOVS). METHODS: This study included 46 patients with pharyngeal cancer who underwent 3D TOVS. The perioperative complications and survival curves were retrospectively analyzed. RESULTS: One patient with oropharyngeal cancer who underwent neck dissection and transoral resection simultaneously experienced postoperative hemorrhage of the neck. Another patient with oropharyngeal cancer underwent hemostasis for postoperative pharyngeal hemorrhage. There was one case of aspiration pneumonia. One patient developed cervical lymph node recurrence; however, there was no local recurrence or primary mortality. The 2-year overall survival, disease-specific survival, local control rates, locoregional control rate, and invasive disease-free survival were 90.9%, 100%, 100%, 97.4%, and 79.9%, respectively. CONCLUSIONS: Three-dimensional endoscopy can be safely applied to TOVS.

Structural and thermodynamic insights into antibody light chain tetramer formation through 3D domain swapping.

Overexpression of antibody light chains in small plasma cell clones can lead to misfolding and aggregation. On the other hand, the formation of amyloid fibrils from antibody light chains is related to amyloidosis. Although aggregation of antibody light chain is an important issue, atomic-level structural examinations of antibody light chain aggregates are sparse. In this study, we present an antibody light chain that maintains an equilibrium between its monomeric and tetrameric states. According to data from X-ray crystallography, thermodynamic and kinetic measurements, as well as theoretical studies, this antibody light chain engages in 3D domain swapping within its variable region. Here, a pair of domain-swapped dimers creates a tetramer through hydrophobic interactions, facilitating the revelation of the domain-swapped structure. The negative cotton effect linked to the ß-sheet structure, observed around 215 nm in the circular dichroism (CD) spectrum of the tetrameric variable region, is more pronounced than that of the monomer. This suggests that the monomer contains less ß-sheet structures and exhibits greater flexibility than the tetramer in solution. These findings not only clarify the domain-swapped structure of the antibody light chain but also contribute to controlling antibody quality and advancing the development of future molecular recognition agents and drugs.

Necrotizing fasciitis wound after debridement could be successfully treated with negative-pressure wound therapy with instillation and dwelling: A case report.

Background: Necrotizing fasciitis (NF) is associated with a high mortality rate. Adequate incision and drainage and repeated debridement are necessary for NF management. After drainage, daily local irrigation should be performed. Case presentation: A 72-year-old male patient complained of left lower quadrant pain. Computed tomography revealed a 7 cm mass in the descending colon, with retroperitoneal penetration. Hence, he underwent emergency surgery. The left abdomen was widely incised, and a transverse colostomy was performed for local wound control. Daily debridement of necrotic tissue and wound irrigation were continued. On postoperative day 48, the wound was extensive and complex and obtained a positive bacterial culture. Subsequently, we began a negative-pressure wound therapy with instillation and dwelling (NPWTi-d), which was very effective for extensive and complicated wounds with infection. Thereafter, a split-thickness skin was grafted, and the skin graft survived well. Ultimately, the wound successfully closed. Conclusions: NPWT is contraindicated for infected wounds, and an infection control period is required. However, NPWTi-d enables early initiation of wound care despite the presence of infection. Therefore, NPWTi-d is effective for extensive and complicated wounds with infection after NF debridement.

Kre6 (yeast 1,6-ß-transglycosylase) homolog, PhTGS, is essential for ß-glucan synthesis in the haptophyte Pleurochrysis haptonemofera.

Haptophytes synthesize unique ß-glucans containing more ß-1,6-linkages than ß-1,3 linkages, as a storage polysaccharide. To understand the mechanism of the synthesis, we investigated the roles of Kre6 (yeast 1,6-ß-transglycosylase) homologs, PhTGS, in the haptophyte Pleurochrysis haptonemofera. RNAi of PhTGS repressed ß-glucan accumulation and simultaneously induced lipid production, suggesting that PhTGS is involved in ß-glucan synthesis and that the knockdown leads to the alteration of the carbon metabolic flow. PhTGS was expressed more in light, where ß-glucan was actively produced by photosynthesis, than in the dark. The crude extract of E. coli expressing PhKre6 demonstrated its activity to incorporate 14C-UDP-glucose into ß-glucan of P. haptonemofera. These findings suggest that PhTGS functions in storage ß-glucan synthesis specifically in light, probably by producing the ß-1,6-branch.

Continuous visualization and validation of pain in critically ill patients using artificial intelligence: a retrospective observational study.

Machine learning tools have demonstrated viability in visualizing pain accurately using vital sign data; however, it remains uncertain whether incorporating individual patient baselines could enhance accuracy. This study aimed to investigate improving the accuracy by incorporating deviations from baseline patient vital signs and the concurrence of the predicted artificial intelligence values with the probability of critical care pain observation tool (CPOT) ≥ 3 after fentanyl administration. The study included adult patients in intensive care who underwent multiple pain-related assessments. We employed a random forest model, utilizing arterial pressure, heart rate, respiratory rate, gender, age, and Richmond Agitation-Sedation Scale score as explanatory variables. Pain was measured as the probability of CPOT scores of ≥ 3, and subsequently adjusted based on each patient's baseline. The study included 10,299 patients with 117,190 CPOT assessments. Of these, 3.3% had CPOT scores of ≥ 3. The random forest model demonstrated strong accuracy with an area under the receiver operating characteristic curve of 0.903. Patients treated with fentanyl were grouped based on CPOT score improvement. Those with ≥ 1-h of improvement after fentanyl administration had a significantly lower pain index (P = 0.020). Therefore, incorporating deviations from baseline patient vital signs improved the accuracy of pain visualization using machine learning techniques.

Diagnostic utility of EUS-FNA in distinguishing hepatocellular carcinoma from gastric submucosal tumor: A case report.

INTRODUCTION AND IMPORTANCE: Extrahepatically growing hepatocellular carcinoma (HCC) account for only 0.3 %-2.4 % of all hepatocellular carcinoma cases. We present the rare case of a patient in whom endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) was used to distinguish an extrahepatically growing HCC from a gastric submucosal tumor. CASE PRESENTATION: The patient was a 44-year-old man who underwent an esophagogastroduodenoscopy (EGD) and was found to have a submucosal tumor (SMT). A gastrointestinal stromal tumor was suspected. Computed tomography (CT) showed an unclear connecting between the tumor and the liver. On immunohistochemical analysis, the tumor cells were positive for CK7, PIVKA-II, and glypican-3, and negative for C-kit. Based on these findings, HCC was suspected, and ethoxybenzyl diethylenetriamine-enhanced magnetic resonance imaging (EOB-MRI) was performed. EOB-MRI revealed the connecting of the tumor and the liver. Thus, the patient was diagnosed as having an extrahepatically protruding HCC, and laparoscopic S2 partial hepatectomy was performed. Pathologically, the tumor was identified as a moderately differentiated HCC. According to the 8th edition of the UICC TNM Classification, the tumor was classified as T1bN0M0 and stage IB. CLINICAL DISCUSSION: Owing to its rarity, the diagnosis of an extrahepatically protruding HCC is often difficult when the differentiation of a gastric SMT and HCC is unclear on CT. As in this case, if an HCC is also differentiated on hematoxylin and eosin staining after EUS-FNA, immunohistochemistry findings can help in the diagnosis. CONCLUSION: The findings show that EUS-FNA could be helpful for diagnosis when an HCC needs to be differentiated from a gastric SMT.

Protein Cages and Nanostructures Constructed from Protein Nanobuilding Blocks.

Protein cages and nanostructures are promising biocompatible medical materials, such as vaccines and drug carriers. Recent advances in designed protein nanocages and nanostructures have opened up cutting-edge applications in the fields of synthetic biology and biopharmaceuticals. A simple approach for constructing self-assembling protein nanocages and nanostructures is the design of a fusion protein composed of two different proteins forming symmetric oligomers. In this chapter, we describe the design and methods of protein nanobuilding blocks (PN-Blocks) using a dimeric de novo protein WA20 to construct self-assembling protein cages and nanostructures. A protein nanobuilding block (PN-Block), WA20-foldon, was developed by fusing an intermolecularly folded dimeric de novo protein WA20 and a trimeric foldon domain from bacteriophage T4 fibritin. The WA20-foldon self-assembled into several oligomeric nanoarchitectures in multiples of 6-mer. De novo extender protein nanobuilding blocks (ePN-Blocks) were also developed by fusing tandemly two WA20 with various linkers, to construct self-assembling cyclized and extended chain-like nanostructures. These PN-Blocks would be useful for the construction of self-assembling protein cages and nanostructures and their potential applications in the future.

Investigation of Risk Factors for Postoperative Delirium after Transcatheter Aortic Valve Implantation: A Retrospective Study.

Transcatheter aortic valve implantation (TAVI) is an effective treatment for severe aortic stenosis (AS); however, postoperative delirium (POD) can worsen patient outcomes. This study aimed to examine the risk factors for POD after TAVI, including possible intervening factors. We included 87 patients (mean age: 83) who underwent TAVI between May 2014 and September 2018. POD was defined by the presence or absence of delirium on ICU admission, assessed using the Confusion Assessment Method for the ICU. Factors that showed significant differences in the univariate analysis were analyzed using a multiple logistic regression analysis. In total, 31 patients (36%) had POD after ICU admission, and 56 (64%) did not. The preoperative frailty score and aortic valve opening area (AVA) were significant risk factors for POD. The multivariate analysis also showed that both factors were independent risk factors for POD (area under the receiver operating characteristic curve: 0.805). There were no significant differences in the number of ICU days. However, postoperative hospitalization was significantly longer in the POD group (19 (17-31) days vs. 16 (13-22) days; p = 0.002). POD was associated with a narrow AVA and frailty; this suggests that frailty prevention interventions according to the AVA may be important.

A case of strongly suspected Lynch syndrome with colorectal neuroendocrine carcinoma.

BACKGROUND: Neuroendocrine cell carcinomas (NEC) of the colon and rectum are uncommon, representing ~ 0.1% of all colorectal carcinomas. They are associated with a much worse prognosis compared to adenocarcinoma of the colon and rectum, as death occurs in approximately half of all patients within 1 year. Lynch syndrome (LS) is the most common cause of inherited colorectal cancer, accounting for 2-4% of newly diagnosed colorectal cancer cases. This case is extremely rare which was strongly suspected LS as the background, and NEC as the histological type of colorectal cancer. CASE PRESENTATION: The patient was a 44-year-old man presenting with vomiting as the main complaint. He had undergone ileocecal resection for cecal cancer at age 29. The diagnosis was obstructive descending colorectal cancer, and colonoscopy revealed tumors in the rectum and sigmoid colon in addition. Due to multiple occurrences of colorectal cancer and its prevalence in the patient's family, LS was suspected. The operation which was a subtotal proctocolectomy was performed. Pathological analysis revealed complete curative resection and the descending colon cancer of the obstructed portion was at the most advanced pathological Stage IIIC in UICC TNM classification, and the tissue type was a NEC. The Ki-67 index was 70%. The results of the microsatellite instability (MSI) test showed high-frequency MSI. The BRAF V600E variant was negative. The immunoexpression of MLH1 was positive, MSH2 was negative, PMS2 was positive, and MSH6 was negative. CONCLUSIONS: Extended surgery is recommended for incipient colorectal cancer in LS cases in order to reliably reduce the risk of developing metachronous colorectal cancer. The survival outcome of surgery alone on digestive tract NECs, even locoregional lesions that are completely resection, is extremely poor. It is currently unclear if digestive tract NECs develop more readily in patients with LS. The accumulation of additional cases is necessary.

Use of 3D domain swapping in constructing supramolecular metalloproteins.

Supramolecules, which are formed by assembling multiple molecules by noncovalent intermolecular interactions instead of covalent bonds, often show additional properties that cannot be exhibited by a single molecule. Supramolecules have evolved into molecular machines in the field of chemistry, and various supramolecular proteins are responsible for life activities in the field of biology. The design and creation of supramolecular proteins will lead to development of new enzymes, functional biomaterials, drug delivery systems, etc.; thus, the number of studies on the regulation of supramolecular proteins is increasing year by year. Several methods, including disulfide bond, metal coordination, and surface-surface interaction, have been utilized to construct supramolecular proteins. In nature, proteins have been shown to form oligomers by 3D domain swapping (3D-DS), a phenomenon in which a structural region is exchanged between molecules of the same protein. We have been studying the mechanism of 3D-DS and utilizing 3D-DS to construct supramolecular metalloproteins. Cytochrome c forms cyclic oligomers and polymers by 3D-DS, whereas other metalloproteins, such as various c-type cytochromes and azurin form small oligomers and myoglobin forms a compact dimer. We have also utilized 3D-DS to construct heterodimers with different active sites, a protein nanocage encapsulating a Zn-SO4 cluster in the internal cavity, and a tetrahedron with a designed building block protein. Protein oligomer formation was controlled for the 3D-DS dimer of a dimer-monomer transition protein. This article reviews our research on supramolecular metalloproteins.

Be careful of splenic rupture caused by hit by a pitch during a baseball game: a case report.

BACKGROUND: To the best of our knowledge, splenic rupture caused by hit by a pitch (HBP) has not been previously reported. We present a patient who underwent emergency laparotomy for splenic rupture after being HBP during a baseball game. CASE PRESENTATION: A 41-year-old male was HBP in the left abdomen during his first at-bat during a baseball game. During the operation, vascular injury of the splenic hilum and a deeply extending parenchymal injury were observed, and splenectomy was performed. Histologic findings were consistent with splenic rupture. CONCLUSIONS: The patient's postoperative course was uneventful. Although extremely rare, the possibility of intra-abdominal organ injury should be considered in batters who are hit in the abdomen by a pitched baseball, as illustrated by our patient.

Semi-automated tracking of pain in critical care patients using artificial intelligence: a retrospective observational study.

Monitoring the pain intensity in critically ill patients is crucial because intense pain can cause adverse events, including poor survival rates; however, continuous pain evaluation is difficult. Vital signs have traditionally been considered ineffective in pain assessment; nevertheless, the use of machine learning may automate pain assessment using vital signs. This retrospective observational study was performed at a university hospital in Sendai, Japan. Objective pain assessments were performed in eligible patients using the Critical-Care Pain Observation Tool (CPOT). Three machine-learning methods-random forest (RF), support vector machine (SVM), and logistic regression (LR)-were employed to predict pain using parameters, such as vital signs, age group, and sedation levels. Prediction accuracy was calculated as the harmonic mean of sensitivity, specificity, and area under the receiver operating characteristic curve (AUROC). Furthermore, 117,190 CPOT assessments were performed in 11,507 eligible patients (median age: 65 years; 58.0% males). We found that pain prediction was possible with all three machine-learning methods. RF demonstrated the highest AUROC for the test data (RF: 0.853, SVM: 0.823, and LR: 0.787). With this method, pain can be objectively, continuously, and semi-automatically evaluated in critically ill patients.

Favorable outcomes of surgical resection for extrahepatic recurrent hepatocellular carcinoma.

AIM: Repeat resection for intrahepatic recurrent hepatocellular carcinoma (HCC) is effective for the long-term survival of patients; however, little is known about the surgical outcomes of extrahepatic nodules. The aim of this study is to investigate whether resection can contribute to the survival of patients with extrahepatic recurrent HCC. METHODS: Under the conditions that intrahepatic recurrent HCC was absent or controlled by locoregional therapies, patients who had resectable extrahepatic recurrent HCC in the lymph nodes, adrenal gland, peritoneum, lung, or brain were included in this study. The survival of patients who did (Surgical group) and did not (Non-surgical group, underwent other therapies) undergo resection for extrahepatic recurrent HCC was compared. RESULTS: Thirty-eight and 26 patients were included in the Surgical and Non-surgical groups, respectively. No patient had severe postoperative complications. After a median follow-up of 1.2 (range, 0.2-8.8) years, the median cumulative incidence of extrahepatic recurrent HCC was 1.2 years (95% confidence interval [CI], 0.4-3.5) in the Surgical group. The median overall survival was 5.3 (95% CI, 2.5-8.8) and 1.1 (0.8-2.3) years in the Surgical and Non-surgical groups, respectively (P < 0.001). The 5-year rates of survival were 60.5% and 9.1% in the Surgical and Non-surgical groups, respectively. Surgical resection, α-fetoprotein, disease-free interval, and metastasis at the adrenal gland were the independent factors for overall survival. CONCLUSIONS: Due to the favorable surgical outcomes, resection should be considered as one of the therapeutic choices for patients with extrahepatic recurrent HCC if intrahepatic recurrent HCC can be controlled by locoregional therapies.

Impact of marginal resection for hepatocellular carcinoma.

PURPOSE: The surgical margin for liver resection to treat hepatocellular carcinoma (HCC) is occasionally < 1 mm. This study determined the impact of a surgical margin < 1 mm [marginal resection (MR)] on the types of recurrence and the prognosis in solitary HCC. METHODS: The data of 454 patients undergoing curative liver resection for solitary HCC in our institution were analyzed. The patients were divided into the MR (n = 90) and non-MR (n = 364) groups. The clinicopathological data and outcomes after liver resection were compared. A case-matching analysis using a propensity scoring method was also performed. RESULTS: The recurrence-free survival was significantly and overall survival was marginally significantly lower in the MR group than in the non-MR group (p = 0.012-0.051, respectively). According to a multivariate analysis, MR was not a significant independent factor for recurrence-free survival (p = 0.056). After propensity score matching, there were no significant differences in the recurrence-free and overall survival between the two groups (p = 0.375-0.496, respectively). Furthermore, there were no significant differences in the intrahepatic recurrence patterns between the two groups before and after matching. CONCLUSION: MR for solitary HCC might be sufficient in patients with a limited liver functional reserve.

Arterial blood pressure correlates with 90-day mortality in sepsis patients: a retrospective multicenter derivation and validation study using high-frequency continuous data.

OBJECTIVE: To identify the outcome of patients with sepsis using high-frequency blood pressure data. MATERIALS AND METHODS: This retrospective observational study was conducted at a university hospital ICU (derivation study) and at two urban hospitals (validation study) with data from adult sepsis patients who visited the centers during the same period. The area under the curve (AUC) of blood pressure falling below threshold was calculated. The predictive 90-day mortality (primary endpoint) area under threshold (AUT) and critical blood pressure were calculated as the maximum area under the curve of the receiver operating characteristic curve (AUCROC) and the threshold minus average AUT (derivation study), respectively. For the validation study, the derived 90-day mortality AUCROC (using critical blood pressure) was compared with Sequential Organ Failure Assessment (SOFA), Simplified Acute Physiology Score (SAPS) II, Acute Physiology and Chronic Health Evaluation (APACHE) II, and APACHE III. RESULTS: Derivation cohort (N = 137): the drop area from the mean blood pressure of 70 mmHg at 24-48 hours most accurately predicted 90-day mortality [critical blood pressure, 67.8 mmHg; AUCROC, 0.763; 95% confidence interval (CI), 0.653-0.890]. Validation cohort (N = 141): the 90-day mortality AUCROC (0.776) compared with the AUCROC for SOFA (0.711), SAPSII (0.771), APACHE II (0.745), and APACHE III (0.710) was not significantly different from the critical blood pressure 67.8 mmHg (P = 0.420). CONCLUSION: High-frequency arterial blood pressure data of the period and extent of blood pressure depression can be useful in predicting the clinical outcomes of patients with sepsis.

Identification of Proteins Differentially Expressed by Adipose-derived Mesenchymal Stem Cells Isolated from Immunodeficient Mice.

Although cell therapy using adipose-derived mesenchymal stem cells (AdMSCs) regulates immunity, the degree to which cell quality and function are affected by differences in immunodeficiency of donors is unknown. We used liquid chromatography tandem-mass spectrometry (LC MS/MS) to identify the proteins expressed by mouse AdMSCs (mAsMSCs) isolated from normal (C57BL/6) mice and mice with severe combined immunodeficiency (SCID). The protein expression profiles of each strain were 98%-100% identical, indicating that the expression levels of major proteins potentially associated with the therapeutic effects of mAdMSCs were highly similar. Further, comparable levels of cell surface markers (CD44, CD90.2) were detected using flow cytometry or LC MS/MS. MYH9, ACTN1, CANX, GPI, TPM1, EPRS, ITGB1, ANXA3, CNN2, MAPK1, PSME2, CTPS1, OTUB1, PSMB6, HMGB1, RPS19, SEC61A1, CTNNB1, GLO1, RPL22, PSMA2, SYNCRIP, PRDX3, SAMHD1, TCAF2, MAPK3, RPS24, and MYO1E, which are associated with immunity, were expressed at higher levels by the SCID mAdMSCs compared with the C57BL/6 mAdMSCs. In contrast, ANXA9, PCBP2, LGALS3, PPP1R14B, and PSMA6, which are also associated with immunity, were more highly expressed by C57BL/6 mAdMSCs than SCID mAdMSCs. These findings implicate these two sets of proteins in the pathogenesis and maintenance of immunodeficiency.

Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors.

We recently demonstrated the generation of mouse induced tissue-specific stem (iTS) cells through transient overexpression of reprogramming factors combined with tissue-specific selection. Here we induced expandable tissue-specific progenitor (iTP) cells from human pancreatic tissue through transient expression of genes encoding the reprogramming factors OCT4 (octamer-binding transcription factor 4), p53 small hairpin RNA (shRNA), SOX2 (sex-determining region Y-box 2), KLF4 (Kruppel-like factor 4), L-MYC, and LIN28. Transfection of episomal plasmid vectors into human pancreatic tissue efficiently generated iTP cells expressing genetic markers of endoderm and pancreatic progenitors. The iTP cells differentiated into insulin-producing cells more efficiently than human induced pluripotent stem cells (iPSCs). iTP cells continued to proliferate faster than pancreatic tissue cells until days 100-120 (passages 15-20). iTP cells subcutaneously inoculated into immunodeficient mice did not form teratomas. Genomic bisulfite nucleotide sequence analysis demonstrated that the OCT4 and NANOG promoters remained partially methylated in iTP cells. We compared the global gene expression profiles of iPSCs, iTP cells, and pancreatic cells (islets >80%). Microarray analyses revealed that the gene expression profiles of iTP cells were similar, but not identical, to those of iPSCs but different from those of pancreatic cells. The generation of human iTP cells may have important implications for the clinical application of stem/progenitor cells.

A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of Primary Cultured Cells and Subcultured Cells Using Mouse Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (MSC-ATs) are representative cell sources for cell therapy. However, how cell stress resulting from passage influences the MSC-AT protein expression has been unclear. In this study, a protein expression analysis was performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) using mouse primary cultured cells (P0) and cells passaged three times (P3) as samples. A total of 256 proteins were classified as cellular process-related proteins, while 179 were classified as metabolic process-related proteins in P0. These were considered to be adaptive responses of the cells to an in vitro environment. However, seven proteins of growth were identified (Csf1, App, Adam15, Alcam, Tbl1xr1, Ninj1, and Sbds) in P0. In addition, four proteins of antioxidant activity were also identified (Srxn1, Txndc17, Fam213b, and Apoe) in P0. We identified 1139 proteins expressed in both P0 and P3 cells that had their expression decreased to 69.4% in P3 cells compared with P0 cells, but 1139 proteins are very likely proteins that are derived from MSC-AT. The function of MSC-ATs was maintained after three passages. However, the LC-MS/MS analysis data showed that the protein expression was degraded after three passages. MSC-ATs retained about 70% of their protein expression ability in P3 cells.