Sex-Dependent Variations in Hypothalamic Fatty Acid Profile and Neuropeptides in Offspring Exposed to Maternal Obesity and High-Fat Diet.

Maternal obesity and/or high-fat diet (HF) consumption can disrupt appetite regulation in their offspring, contributing to transgenerational obesity and metabolic diseases. As fatty acids (FAs) play a role in appetite regulation, we investigated the maternal and fetal levels of FAs as potential contributors to programmed hyperphagia observed in the offspring of obese dams. Female mice were fed either a control diet (CT) or HF prior to mating, and fetal and maternal blood and tissues were collected at 19 days of gestation. Elevated levels of linoleic acid were observed in the serum of HF dams as well as in the serum of their fetuses. An increased concentration of eicosadienoic acid was also detected in the hypothalamus of female HF-O fetuses. HF-O male fetuses showed increased hypothalamic neuropeptide Y (Npy) gene expression, while HF-O female fetuses showed decreased hypothalamic pro-opiomelanocortin (POMC) protein content. Both male and female fetuses exhibited reduced hypothalamic neurogenin 3 (NGN-3) gene expression. In vitro experiments confirmed that LA contributed to the decreased gene expression of Pomc and Ngn-3 in neuronal cells. During lactation, HF female offspring consumed more milk and had a higher body weight compared to CT. In summary, this study demonstrated that exposure to HF prior to and during gestation alters the FA composition in maternal serum and fetal serum and hypothalamus, particularly increasing n-6, which may play a role in the switch from POMC to NPY neurons, leading to increased weight gain in the offspring during lactation.

Semi-Blind Receivers for Two-Hop MIMO Relay Systems with a Combined TSTF-MSMKron Coding.

Due to the increase in the number of mobile stations in recent years, cooperative relaying systems have emerged as a promising technique for improving the quality of fifth-generation (5G) wireless networks with an extension of the coverage area. In this paper, we propose a two-hop orthogonal frequency division multiplexing and code-division multiple-access (OFDM-CDMA) multiple-input multiple-output (MIMO) relay system, which combines, both at the source and relay nodes, a tensor space-time-frequency (TSTF) coding with a multiple symbol matrices Kronecker product (MSMKron), called TSTF-MSMKron coding, aiming to increase the diversity gain. It is first established that the signals received at the relay and the destination satisfy generalized Tucker models whose core tensors are the coding tensors. Assuming the coding tensors are known at both nodes, tensor models are exploited to derive two semi-blind receivers, composed of two steps, to jointly estimate symbol matrices and individual channels. Necessary conditions for parameter identifiability with each receiver are established. Extensive Monte Carlo simulation results are provided to show the impact of design parameters on the symbol error rate (SER) performance, using the zero-forcing (ZF) receiver. Next, Monte Carlo simulations illustrate the effectiveness of the proposed TSTF-MSMKron coding and semi-blind receivers, highlighting the benefit of exploiting the new coding to increase the diversity gain.

Enhancing decision-making support by mining social media data with social network analysis.

This paper explores the use of social network analysis (SNA) on airlines' online social networks (OSNs) to extract valuable information for decision support, by analyzing interactions and discursive exchanges between users. The research is focused on fostering customer service of an airline company during a strike period, namely by detecting influential customers (whether satisfied or dissatisfied), address pending requests, and enhancing customer satisfaction, thus promoting issue-solving, and increasing responsiveness. The methodology involves analyzing data from the Facebook account of an airline company, using SNA to structure the data, and calculating metrics to detect possible situations to be addressed by customer service. The research concludes that it is possible to extract valuable information for decision support by analyzing the metrics that were built over the interactions and discursive exchanges between OSN users. SNA metrics enable to measure airline's call-center performance in terms of speed of answer and customer satisfaction, to identify active users requiring additional support, as well as highly influential customers who may impact on the overall customer satisfaction, thus helping to resolve issues more efficiently. This study provides both theoretical and practical implications: it contributes to the existing literature by integrating social interaction and SNA for decision support in airline's service context; and it provides practical insights into how companies can use SNA metrics to improve customer service. The research also highlights and corroborates the importance of monitoring social media interactions for decision-making and improving customer service.

Partial freezing of rat livers extends preservation time by 5-fold.

The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between -4 and -6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (-10 to -15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.

Multi-Sensor Wearable Health Device Framework for Real-Time Monitoring of Elderly Patients Using a Mobile Application and High-Resolution Parameter Estimation.

Automatized scalable healthcare support solutions allow real-time 24/7 health monitoring of patients, prioritizing medical treatment according to health conditions, reducing medical appointments in clinics and hospitals, and enabling easy exchange of information among healthcare professionals. With recent health safety guidelines due to the COVID-19 pandemic, protecting the elderly has become imperative. However, state-of-the-art health wearable device platforms present limitations in hardware, parameter estimation algorithms, and software architecture. This paper proposes a complete framework for health systems composed of multi-sensor wearable health devices (MWHD), high-resolution parameter estimation, and real-time monitoring applications. The framework is appropriate for real-time monitoring of elderly patients' health without physical contact with healthcare professionals, maintaining safety standards. The hardware includes sensors for monitoring steps, pulse oximetry, heart rate (HR), and temperature using low-power wireless communication. In terms of parameter estimation, the embedded circuit uses high-resolution signal processing algorithms that result in an improved measure of the HR. The proposed high-resolution signal processing-based approach outperforms state-of-the-art HR estimation measurements using the photoplethysmography (PPG) sensor.

Error-Robust Distributed Denial of Service Attack Detection Based on an Average Common Feature Extraction Technique.

In recent years, advanced threats against Cyber-Physical Systems (CPSs), such as Distributed Denial of Service (DDoS) attacks, are increasing. Furthermore, traditional machine learning-based intrusion detection systems (IDSs) often fail to efficiently detect such attacks when corrupted datasets are used for IDS training. To face these challenges, this paper proposes a novel error-robust multidimensional technique for DDoS attack detection. By applying the well-known Higher Order Singular Value Decomposition (HOSVD), initially, the average value of the common features among instances is filtered out from the dataset. Next, the filtered data are forwarded to machine learning classification algorithms in which traffic information is classified as a legitimate or a DDoS attack. In terms of results, the proposed scheme outperforms traditional low-rank approximation techniques, presenting an accuracy of 98.94%, detection rate of 97.70% and false alarm rate of 4.35% for a dataset corruption level of 30% with a random forest algorithm applied for classification. In addition, for error-free conditions, it is found that the proposed approach outperforms other related works, showing accuracy, detection rate and false alarm rate of 99.87%, 99.86% and 0.16%, respectively, for the gradient boosting classifier.

Data Security and Trading Framework for Smart Grids in Neighborhood Area Networks.

Due to the drastic increase of electricity prosumers, i.e., energy consumers that are also producers, smart grids have become a key solution for electricity infrastructure. In smart grids, one of the most crucial requirements is the privacy of the final users. The vast majority of the literature addresses the privacy issue by providing ways of hiding user's electricity consumption. However, open issues in the literature related to the privacy of the electricity producers still remain. In this paper, we propose a framework that preserves the secrecy of prosumers' identities and provides protection against the traffic analysis attack in a competitive market for energy trade in a Neighborhood Area Network (NAN). In addition, the amount of bidders and of successful bids are hidden from malicious attackers by our framework. Due to the need for small data throughput for the bidders, the communication links of our framework are based on a proprietary communication system. Still, in terms of data security, we adopt the Advanced Encryption Standard (AES) 128 bit with Exclusive-OR (XOR) keys due to their reduced computational complexity, allowing fast processing. Our framework outperforms the state-of-the-art solutions in terms of privacy protection and trading flexibility in a prosumer-to-prosumer design.

Factors Associated with Recurrence in Takotsubo Syndrome: A Systematic Review / Fatores Associados à Recorrência na Síndrome de Takotsubo: Uma Revisão Sistemática

Abstract Background: Takotsubo syndrome (TTS) is characterized by a temporary systolic dysfunction of the left ventricle (LV) related to a stressful event. However, the factors associated with its recurrence are still not well established. Objective: To analyze the main factors associated with TTS recurrence. Methods: A systematic review was performed using the PRISMA model. Observational studies, published between January 2008 and October 2017, which presented a recurrence rate of at least 3% and/or 5 or more patients with recurrence, and who met at least 80% of the STROBE criteria were included. Results: six articles reached the criteria to compose this systematic review. The recurrence rate ranged from 1 to 3.5% per year (global recurrence rate 3.8%). One study associated higher recurrence rate with the female gender, four reported the time between the first and second episodes, one study associated body mass index (BMI) and hypercontractility of the LV middle anterior wall to a higher recurrence rate. No association between recurrence and electrocardiographic changes were determined. Beta-blockers use was not associated with recurrence rates. Conclusions: Female gender, time from the first episode of the syndrome, low BMI and midventricular obstruction were reported as potential predictors of TTS recurrence.

Resumo Fundamento: A síndrome de Takotsubo (STT) é caracterizada por uma disfunção sistólica temporária do ventrículo esquerdo (VE) relacionada a um evento estressante. No entanto, os fatores associados à sua recorrência ainda não estão bem estabelecidos. Objetivo: Analisar os principais fatores associados à recorrência da STT. Métodos: Uma revisão sistemática foi realizada usando o modelo PRISMA. Foram incluídos estudos observacionais, publicados entre janeiro de 2008 e outubro de 2017, que apresentaram uma taxa de recorrência de pelo menos 3% e/ou 5 ou mais pacientes com recidiva e que preencheram pelo menos 80% dos critérios STROBE. Resultados: Seis artigos atenderam aos critérios para esta revisão sistemática. A taxa de recorrência variou de 1 a 3,5% ao ano (taxa de recorrência global 3,8%). Um estudo associou maior taxa de recorrência ao sexo feminino, quatro relataram o tempo entre o primeiro e o segundo episódio, um estudo associou o índice de massa corporal (IMC) e a hipercontratilidade da parede anterior média do VE a uma maior taxa de recorrência. Não foi determinada associação entre recorrência e alterações eletrocardiográficas. O uso de betabloqueadores não foi associado a taxas de recorrência. Conclusões: Sexo feminino, tempo desde o primeiro episódio da síndrome, baixo IMC e obstrução ventricular foram relatados como possíveis preditores de recorrência da STT.

Factors Associated with Recurrence in Takotsubo Syndrome: A Systematic Review.

BACKGROUND: Takotsubo syndrome (TTS) is characterized by a temporary systolic dysfunction of the left ventricle (LV) related to a stressful event. However, the factors associated with its recurrence are still not well established. OBJECTIVE: To analyze the main factors associated with TTS recurrence. METHODS: A systematic review was performed using the PRISMA model. Observational studies, published between January 2008 and October 2017, which presented a recurrence rate of at least 3% and/or 5 or more patients with recurrence, and who met at least 80% of the STROBE criteria were included. RESULTS: six articles reached the criteria to compose this systematic review. The recurrence rate ranged from 1 to 3.5% per year (global recurrence rate 3.8%). One study associated higher recurrence rate with the female gender, four reported the time between the first and second episodes, one study associated body mass index (BMI) and hypercontractility of the LV middle anterior wall to a higher recurrence rate. No association between recurrence and electrocardiographic changes were determined. Beta-blockers use was not associated with recurrence rates. CONCLUSIONS: Female gender, time from the first episode of the syndrome, low BMI and midventricular obstruction were reported as potential predictors of TTS recurrence.

Tensor-Based Subspace Tracking for Time-Delay Estimation in GNSS Multi-Antenna Receivers.

Although Global Navigation Satellite Systems (GNSS) receivers currently achieve high accuracy when processing their geographic location under line of sight (LOS), multipath interference and noise degrades the accuracy considerably. In order to mitigate multipath interference, receivers based on multiple antennas became the focus of research and technological development. In this context, tensor-based approaches based on Parallel Factor Analysis (PARAFAC) models have been proposed in the literature, providing optimum performance. State-of-the-art techniques for antenna array based GNSS receivers compute singular value decomposition (SVD) for each new sample, implying into a high computational complexity, being, therefore, prohibitive for real-time applications. Therefore, in order to reduce the computational complexity of the parameter estimates, subspace tracking algorithms are essential. In this work, we propose a tensor-based subspace tracking framework to reduce the overall computational complexity of the highly accurate tensor-based time-delay estimation process.

The Role of the LY294002 - A Non-Selective Inhibitor of Phosphatidylinositol 3-Kinase (PI3K) Pathway- in Cell Survival and Proliferation in Cell Line SCC-25.

The activation of PI3K further activates subsequent regulatory pathways, which are activated via AKT phosphorylation. AKT is closely related to the Bcl-2 family, a protein known to be involved in cell survival. AKT also has a relationship with inflammatory and glycolytic mediators. The present work aimed to evaluate the relationship between the PI3K/AKT pathway, cell survival/proliferation, inflammatory mediators and the glycolytic pathway in oral squamous cell carcinoma. All experiments were performed in the SCC25 oral squamous cell carcinoma cell line. In the presence or absence of PI3K pathway inhibitors, we analyzed the protein expression of pAKT and AKT; X-linked inhibitor of apoptosis protein; Bcl-2-associated death promoter; Bcl-2-like protein two inhibitor; cyclooxygenase 1; cyclooxygenase-2; and glycoprotein-associated glucose transporter 1. For the functional characterization of treated or untreated cells, we also performed matrix invasion assays, cell migration assays, and cell proliferation assays. Our results demonstrated that activation of the PI3K/AKT pathway is directly related to members of the Bcl-2 family and GLUT1, but not the inflammatory mediators COX1 and COX2. Our data suggest that the PI3K/AKT pathway is related to cell survival and proliferation in oral squamous cell carcinoma through its interaction with Bcl-2 family members.
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Does virginiamycin supplementation affect the metabolism and performance of Nellore bulls grazing under low and high gain rates?

This study aimed to evaluate the effect of virginiamycin on the metabolism and performance of growing Nellore bulls under low and high gain rates on pasture. In experiment 1, 80 Nellore bulls (age = 12 ± 2 months, body weight = 258 ± 15 kg) were assigned to 16 paddocks in a 2 × 2 randomized block factorial arrangement. In experiment 2, 12 cannulated Nellore bulls were assigned to three 4 × 4 balanced Latin squares. The factors were: (1) mineral salt without or with virginiamycin, and (2) low or high gain rate. No interaction was noted between factors (p > .10). Animals fed virginiamycin had greater average daily gain (14%, p < .01), body weight (11 kg, p = .05), plasma nonesterified fatty acid (20%, p < .01), serum calcium concentration (2.62%, p = .04), and total protozoa (p = .03) and had the same bacterial proportion (p > .27). Animals with a low gain rate had greater serum urea concentration (19.6%, p < .01) and ruminal ammonia nitrogen (62%, p < .01). Thus, virginiamycin increases the performance and changes the metabolism of growing Nellore bulls under low and high gain rates on pasture.

Blood-based biomarkers for the diagnosis and monitoring of gliomas.

Monitoring patient response to treatment is challenging for most cancers, but it is particularly difficult in glioblastoma multiform, the most common and aggressive form of malignant brain tumor. These tumors exhibit a high degree of heterogeneity which may not be reflected in a biopsy. To determine if the current standard of care is effective, glioma patients are monitored using MRI or CT scans, an effective but sometimes misleading approach due to the phenomenon of pseudoprogression. As such, there is incredible need for a minimally invasive "liquid biopsy" to assist in molecularly characterizing the tumors while also aiding in the identification of true progression in glioblastoma. This review details the status and potential impact for circulating tumor cells, extracellular vesicles, ctDNA, and ctRNA, putative circulating biomarkers found in the blood in glioblastoma patients. As mutation-based therapy becomes more prevalent in gliomas, blood-based analyses may offer a non-invasive method of identifying mutations. The ability to obtain serial "liquid biopsies" will provide unique opportunities to study the evolution of tumors and mechanisms of treatment resistance and monitor for mutational changes in response to therapy.

Electrochemical immunosensor based on ZnO nanorods-Au nanoparticles nanohybrids for ovarian cancer antigen CA-125 detection.

In this research, ZnO nanorods - Au nanoparticles nanohybrids have been fabricated and employed to sensitive electrochemical strategy for the specific detection of the ovarian cancer antigen CA-125/MUC126. The microdevice was developed in our lab based on gold and silver electrodes sputtered on glass substrate. The ZnO nanorods arrays were grown on working electrode using assisted microwave hydrothermal synthesis than gold nanoparticles (Au NPs) were deposited by sputtering. The Au NPs onto ZnO nanorods surface provides a favorable platform for efficient loading of anti-CA-125 antibody via binding with cystamine and glutaraldehyde. The effective loading of the biological material (CA-125 antibody and antigen) on the matrix was observed by SEM images. The electrochemical immunosensor shows a sensitive response to ovarian cancer antigen recombinant human CA-125/MUC126 with detection of 2.5ng/µL, 100 times lower than immunoblot system. Due to high specificity, reproducibility and noteworthy stability, the developed sensor will provide a sensitive, selective and convenient approach to be used to detect CA-125/MUC126.

Gene expression patterns through oral squamous cell carcinoma development: PD-L1 expression in primary tumor and circulating tumor cells.

Oral squamous cell carcinoma (OSCC) is the most common tumor of the oral cavity and has been associated with poor prognosis. Scarce prognostic markers are available for guiding treatment and/or sub-classifying patients. This study aims to identify biomarkers by searching for genes whose expression is increased or decreased during tumor progression (through T1 to T4 stages). Thirty-six samples from all tumor size stages (from T1 to T4) were analyzed using cDNA microarrays. Selected targets were analyzed by immunohistochemistry and in circulating tumor cells by immunofluorescence and Nanostring. Correlation was shown between PD-L1 and tumor size and lymph node metastasis, HOXB9 and tumor size, BLNK and perineural invasion, and between ZNF813 and perineural invasion. PD-L1 positivity was an independent prognostic factor in this cohort (p = 0.044, HH = 0.426). In CTCs from patients with locally advanced OSCC, we found a strong cytoplasmatic expression of PD-L1. PD-L1 is a ligand of PD-1 and is believed to limit T cell activity in inflammatory responses and limit autoimmune diseases. We demonstrated an important role for PD-L1 in primary tumors according to tumor size, and in disease specific survival. Therefore, we could further determine individuals with PD-L1+ CTCs, and possibly follow treatment using CTCs.

Use of local intelligence to reduce energy consumption of wireless sensor nodes in elderly health monitoring systems.

The percentage of elderly people in European countries is increasing. Such conjuncture affects socio-economic structures and creates demands for resourceful solutions, such as Ambient Assisted Living (AAL), which is a possible methodology to foster health care for elderly people. In this context, sensor-based devices play a leading role in surveying, e.g., health conditions of elderly people, to alert care personnel in case of an incident. However, the adoption of such devices strongly depends on the comfort of wearing the devices. In most cases, the bottleneck is the battery lifetime, which impacts the effectiveness of the system. In this paper we propose an approach to reduce the energy consumption of sensors' by use of local sensors' intelligence. By increasing the intelligence of the sensor node, a substantial decrease in the necessary communication payload can be achieved. The results show a significant potential to preserve energy and decrease the actual size of the sensor device units.

CD44+/CD133+ immunophenotype and matrix metalloproteinase-9: Influence on prognosis in early-stage oral squamous cell carcinoma.

BACKGROUND: The purpose of this study was to investigate the expression of CD44 and/or CD133 immunophenotypes and the associated effects of matrix metalloproteinase-9 (MMP-9) in early-stage oral squamous cell carcinomas (SCC) to assess their influence on tumor prognosis. METHODS: The following data were derived from 150 patients: age, sex, primary anatomic site, smoking status, alcohol intake, recurrence, metastases, histological classification, treatment, disease-free survival (DFS), and overall survival (OS). Immunohistochemical study of CD44, CD133, and MMP-9 expression was performed on a tissue microarray of 150 paraffin blocks of oral SCCs. RESULTS: The predominant immunophenotype identified to exhibit a significant correlation with MMP-9 was the CD44+/CD133+. Multivariate analyses identified a significant correlation of OS with surgical treatment and with CD44+/CD133+ immunophenotype. CONCLUSION: This investigation demonstrated the prognostic importance of CD44/CD133 expression, which can help improve the prognostic value of surgical treatment for oral SCCs when diagnosed in early stages.

BRCA1 and γH2AX as independent prognostic markers in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCC) are believed to originate from sequential mutations that can develop as a consequence of genetic instability acquired over time. BRCA1 are linked to DNA recombination and repair processes, being of importance for its role in regulation of RAD51 and H2AX (γH2AX). The aim of this study was to investigate the relationship between BRCA1 expression status and evaluate its prognostic impact. We selected from 150 OSCC patients, and evaluated BRCA1 expression in OSCC by immunohistochemistry and qRT-PCR, comparing its expression with homologous recombination markers (RAD51, γH2AX and p53), clinicopathological and survival data. Expression of BRCA1 was observed in 61 cases (43.88%) and was related to tumor size (T stage) (p=0.001), and gender (p=0.017). mRNA from BRCA1 showed a borderline relationship with perineural invasion (p=0.053). BRCA1 [p=0.030; HR: 2.334 (C.I.: 1.087-5.012)], γH2AX [p=0.045; HR: 0.467 (C.I.: 0.222-0.628)] and gender [p=0.001; HR: 10.386 [(C.I.: 2.679-10.623)] were independent prognostic factors for DSS. BRCA1 and γH2AX expression by OSCC cells are associated with reduced overall survival time, independent of other variables in patients, as well as gender, and our findings shed some light about DSB markers in OSCC and its role as prognostic factors.

The relationship between lymphatic vascular density and vascular endothelial growth factor A (VEGF-A) expression with clinical-pathological features and survival in pancreatic adenocarcinomas.

BACKGROUND: Pancreatic cancer is a rare tumor with an extremely low survival rate. Its known risk factors include the chronic use of tobacco and excessive alcohol consumption and the presence of chronic inflammatory diseases, such as pancreatitis and type 2 diabetes. Angiogenesis and lymphangiogenesis, which have been the focus of recent research, are considered prognostic factors for cancer development. Knowing the angiogenic and lymphangiogenic profiles of a tumor may provide new insights for designing treatments according to the different properties of the tumor. The aim of this study was to evaluate the density of blood and lymphatic vessels, and the expression of VEGF-A, in pancreatic adenocarcinomas, as well as the relationship between blood and lymphatic vascular density and the prognostically important clinical-pathological features of pancreatic tumors. METHODS: Paraffin blocks containing tumor samples from 100 patients who were diagnosed with pancreatic cancer between 1990 and 2010 were used to construct a tissue microarray. VEGF expression was assessed in these samples by immunohistochemistry. To assess the lymphatic and vascular properties of the tumors, 63 cases that contained sufficient material were sectioned routinely. The sections were then stained with the D2-40 antibody to identify the lymphatic vessels and with a CD34 antibody to identify the blood vessels. The vessels were counted individually with the Leica Application Suite v4 program. All statistical analyses were performed using SPSS 18.0 (Chicago, IL, USA) software, and p values ≤ 0.05 were considered significant. RESULTS: In the Cox regression analysis, advanced age (p=0.03) and a history of type 2 diabetes (p=0.014) or chronic pancreatitis (p=0.02) were shown to be prognostic factors for pancreatic cancer. Blood vessel density (BVD) had no relationship with clinical-pathological features or death. Lymphatic vessel density (LVD) was inversely correlated with death (p=0.002), and by Kaplan-Meyer survival analysis, we found a significant association between low LVD (p=0.021), VEGF expression (p=0.023) and low patient survival. CONCLUSIONS: Pancreatic carcinogenesis is related to a history of chronic inflammatory processes, such as type 2 diabetes and chronic pancreatitis. In pancreatic cancer development, lymphangiogenesis can be considered an early event that enables the dissemination of metastases. VEGF expression and low LVD can be considered as poor prognostic factors as tumors with this profile are fast growing and highly aggressive. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5113892881028514.