Co-encapsulation of granzyme B and perforin in nanocapsules for tumour therapy: biomimicking immune cells.

Granzyme B (GrB)-based immunotherapy is of interest for cancer treatment. However, insufficient cellular uptake and a lack of targeting remain challenges to make use of GrB for solid tumour therapy. As GrB induced cell death requires the help of perforin (PFN), we designed a system (nGPM) for the co-delivery of GrB and PFN. Therefore, GrB and PFN were loaded in a porous polymeric nanocapsule rich in acetylcholine analogues and matrix metalloproteinase-2 (MMP-2) responsive peptides. The neutrally charged nGPM nanocapsules showed as long circulating time and accumulated at the tumour sites. Once in the tumour the outside shell of nanocapsules became degraded by overexpressed MMP-2 proteases, resulting in the release of GrB and PFN. We found that the PFN complex formed small pores on the surface of tumour cells which allow GrB to enter the cytoplasm of tumour cells inducing cell apoptosis and tumour suppression significantly.

Observation of the Short-term Efficacy of Technetium-99 Conjugated with Methylene Diphosphonate Combined Therapy in the Treatment of Postmenopausal Osteoporosis.

OBJECTIVE: To investigate the short-term efficacy and safety of Yunke (technetium-99 conjugated methylene diphosphonate) combined with pulsed electromagnetic field (PEMF) and Gukang capsule in the treatment of postmenopausal osteoporosis (PMOP). METHODS: A total of 112 patients with PMOP who received treatment in the Department of Nuclear Medicine of the hospital from January 2019 to June 2020 were selected and randomly divided into 4 groups of 28 patients each. Group A received Yunke and PEMFs, group B received Gukang capsules and PEMFs, group C received Yunke and Gukang capsules and PEMFs, and group D received PEMFs. All groups were given adequate amounts of calcium and active vitamin D. Intervention 2 sessions of 3 months each. Outcome measures were bone mineral density (BMD) and pain improvement. RESULTS: Compared with 1 course of treatment, the symptoms of bone pain were relieved more significantly after 2 courses of treatment in group A (50.0% vs. 64.3%), group B (46.4% vs 64.3%), group C (78.6% vs 92.9%) and group D (21.4% vs 28.6%) (P < 0.05). After 2 courses of treatment, bone pain symptoms were less relieved in group A (96.4% vs. 64.3%), group B (96.4% vs 64.3%), and group D (96.4% vs 28.6%) compared with group C (P < 0.05). Compared with group C, BMD values of L4 vertebrae and femoral neck were significantly decreased in groups A, B, and D (P < 0.05). Compared with those before treatment, BMD of L4 vertebrae and femoral neck increased significantly in groups A, B, C, and D after 2 courses of treatment (P < 0.05). CONCLUSION: Yunke combined therapy can effectively relieve the pain symptoms, increase BMD, and reduce the risk of fracture in patients with PMOP in a short period, which is an effective method for the treatment of PMOP.

TiO2 nanorod arrays@PDA/Ag with biomimetic polydopamine as binary mediators for duplex SERS detection of illegal food dyes.

Based on TiO2 nanorod arrays@PDA/Ag (TNRs@PDA/Ag), a better surface-enhanced Raman scattering (SERS) sensor with effective enrichment and enhancement was investigated for duplex SERS detection of illicit food dyes. Biomimetic PDA functions as binary mediators by utilizing the structural characteristics of polydopamine (PDA), which include the conjugated structure and abundant hydrophilic groups. One PDA functioned as an electron transfer mediator to enhance the efficiency of electron transfer, and the other as an enrichment mediator to effectively enrich rhodamine B (RhB) and crystal violet (CV) through hydrogen bonding, π-π stacking, and electrostatic interactions. Individual and duplex detection of illicit food dyes (RhB and CV) was performed using TNRs@PDA/Ag to estimate SERS applications. Their linear equations and limits of detection of 1 nM for RhB and 5 nM for CV were derived. Individual and duplex food colour detection was successfully accomplished even in genuine chili meal with good results. The bifunctional TNRs@PDA/Ag-based highly sensitive and duplex SERS dye detection will have enormous potential for food safety monitoring.

The impact of air pollution on hospitalization for COPD patients in China.

BACKGROUND: With the rapid development of the global economy and the acceleration of urbanization, air pollution has become a major environmental problem threatening human health. There is limited evidence on the acute effects of air pollution on chronic obstructive pulmonary disease (COPD). METHODS: From 2014 to 2019, we collected data on daily admissions for COPD patients from a city in China. We used the generalized additive model together with distributed lag models to fit the associations of air pollutants with hospital admissions. RESULTS: We observed significant increments in the number of daily admissions (0.086-0.109%) for COPD for a unit range increase in air quality index, PM2.5 and PM10 over four lag days. The impact of air pollution on the number of daily admissions was mainly reflected in the COPD patients who were hospitalized through outpatient departments and tertiary hospitals. DISCUSSION: Short-term exposure to outdoor air pollution may induce the occurrence or exacerbation of COPD patients; therefore, government departments should strengthen the management of air pollution, improve supervision and control mechanisms, pay attention to the quality of medical services, and reduce the adverse effects of air pollution on patients' health.

Generation of GHz line-spacing tunable optical frequency combs using Talbot effects.

In this paper, the generation of GHz line-spacing tunable optical frequency combs (OFCs) was demonstrated using an electro-optical (EO) Talbot laser and a phase modulator. In the EO Talbot laser, the frequency shifting was realized with a dual-parallel Mach-Zehnder modulator (DPMZM) working for carrier-suppressed single-sideband modulation. The PM was employed to achieve the spectral Talbot effect and compensate the phase introduced by the temporal Talbot effect in the laser loop. Arbitrary control of OFC line-spacing was realized using temporal and spectral Talbot effects. The principle of this OFC generator was theoretically modeled. In the experiments, the 2 GHz line spacing of an OFC was multiplied to be 4 GHz, 6 GHz, 8 GHz, and 10 GHz. The frequency spacing of the OFC can also be multiplied with a fractional factor of 3/4, 7/2, 8/5, and 10/7, which was confirmed by simulations.

ST-segment elevation predicts the occurrence of malignant ventricular arrhythmia events in patients with acute ST-segment elevation myocardial infarction.

BACKGROUND: ST-segment elevation (STE) represents a repolarization dispersion marker underlying arrhythmogenesis in ST-segment elevation myocardial infarction (STEMI); however, its value for predicting malignant ventricular arrhythmia events (MVAEs) remains uncertain. METHODS: In total, 285 patients with STEMI and those with or without MVAEs who presented within 6 h of symptom onset were enrolled. The relationships between STE and clinical characteristics of MVAEs (defined as ventricular tachycardia or ventricular fibrillation) were analyzed using t-test, chi-square test, binary multivariate logistic regression, and receiver operating characteristic curve analysis. RESULTS: Patients with STEMI and MVAEs had a shorter time from symptom onset to balloon time (p = 0.0285) and greater STE (p < 0.01) than those without MVAEs. The symptom-to-balloon time, age, and STE were associated with MVAEs after stepwise regression analysis in all cases. Only STE was significantly associated with the occurrence of MVAEs (all, p < 0.01). The area under the curve (AUC) of STE for predicting MVAEs was 0.905, and the cut-off value was 4.5 mV. When only infarct-related arteries were included in the analysis, the AUC of the left anterior descending artery was 0.925 with a cut-off value of 4.5 mV, that of the right coronary artery was 0.915 with a cut-off value of 4.5 mV, and that of the left circumflex artery was 0.929 with a cut-off value of 4.0 mV. CONCLUSIONS: In patients with STEMI presenting within 6 h of symptom onset, age, symptom-to-balloon time, and STE were the main predictors for MVAEs. However, among these, STE was the strongest predictor for MVAEs and was an index for repolarization dispersion of cardiomyocytes in infarcted and non-infarcted areas.

Activation of the IL-17/TRAF6/NF-κB pathway is implicated in Aß-induced neurotoxicity.

BACKGROUND: Neuroinflammation plays a critical role in Amyloid-ß (Aß) pathophysiology. The cytokine, interleukin-17A (IL-17) is involved in the learning and memory process in the central nervous system and its level was reported to be increased in Alzheimer's disease (AD) brain, while the effect of IL-17 on the course of Aß has not been well defined. METHODS: Here, we used APP/PS1 mice to detect the IL-17 expression level. Primary hippocampal neurons were treated with IL-17, and immunofluorescence was used to investigate whether IL-17 induced neuron damage. At the same time, male C57BL/6 mice were injected with Aß42 to mimic the Aß model. Then IL-17 neutralizing antibody (IL-17Ab) was used to inject into the lateral ventricle, and the Open field test, Novel Objective Recognition test, Fear condition test were used to detect cognitive function. LTP was used to assess synaptic plasticity, molecular biology technology was used to assess the IL-17/TRAF6/NF-κB pathway, and ELISA was used to detect inflammatory factors. RESULTS: Altogether, we here found that IL-17 was increased in APP/PS1 mice, and it induced neural damage by the administration to primary hippocampal neurons. Interestingly, Using Aß42 mice, the results showed that the level of IL-17 was increased in Aß42 model mice, and IL-17Ab could ameliorate Aß-induced neurotoxicity and cognitive decline in C57BL/6 mice by downregulation the TRAF6/NF-κB pathway. CONCLUSION: These findings highlight the pathogenic role of IL-17 in Aß induced-synaptic dysfunction and cognitive deficits. Inhibition of IL-17 could ameliorate Aß-induced neurotoxicity and cognitive decline in C57BL/6 mice by downregulation of the TRAF6/NF-κB pathway, which provides new clues for the mechanism of Aß-induced cognitive impairments, and a basis for therapeutic intervention.

Effects of Cadmium on Kidney Function of the Freshwater Turtles Mauremys reevesii.

This research studied the effects of cadmium on kidney function of the freshwater turtles Mauremys reevesii. Turtles were injected intraperitoneally with 0, 7.5, 15, and 30 mg kg-1 cadmium separately for once. The samples were gathered to check the kidney index, the contents of TP in kidney tissue, and the levels of CRE and BUN in the plasma of the turtles. Results showed that the concentration of TP was overall decreased with the extension of cadmium exposure time and the increasing of the exposure dose of cadmium. The CRE content in the plasma of each treatment group increased with the prolongation of exposure time in a dose-dependent, and the BUN levels of all poisoned groups showed a trend of increasing. The kidney index of treated turtles increased. In summary, cadmium could induce the increase of turtle kidney index, the content of CRE and BUN in plasma, and the decrease of TP content in the kidney.

Comprehensive Analysis of N6-Methyladenosine RNA Methylation Regulators in the Diagnosis and Subtype Classification of Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is still a huge danger to human health. Sensitive markers are necessary for the prediction of the risk of AMI and would be beneficial for managing the incidence rate. N6-methyladenosine (m6A) RNA methylation regulators have been confirmed to be involved in the development of various diseases. However, their function in AMI has not been fully elucidated. The purpose of this study was to determine the expression of m6A RNA methylation regulators in AMI as well as their possible functions and prognostic values. The GEO database was used to get the gene expression profiles of patients with and without AMI, and bioinformatics assays of genes with differently expressed expression were performed. We establish two separate m6A subtypes, and relationships between subtypes and immunity were studied. In this study, we identified IGF2BP1, FTO, RBM15, METTL3, YTHDC2, FMR1, and HNRNPA2B1 as the seven major m6A regulators. A nomogram model was developed and confirmed. The consensus clustering algorithm was conducted to categorize AMI patients into two m6A subtypes from the identified m6A regulators. Patients who have activated T-cell activities were found to be in clusterA; they may have a better prognosis as a result. Importantly, we found that patients with high METTL3 expressions had an increased level of Activated.CD4.T.cell and Type.2.T.helper.cell, while having a decreased level of CD56bright.natural.killer.cell, Macrophage, Monocyte, Natural.killer.cell, and Type.17.T.helper.cell. Overall, a diagnostic model of AMI was established based on the genes of IGF2BP1, FTO, RBM15, METTL3, YTHDC2, FMR1, and HNRNPA2B1. Our investigation of m6A subtypes may prove useful in the developments of therapy approaches for AMI.

Stability improvement of a dual-loop optoelectronic oscillator based on self-phase locking.

In this study, we experimentally demonstrate what we believe is a novel dual-loop optoelectronic oscillator (OEO) with a self-phase locking loop (SPLL). Using this feedback loop, the phase of the oscillating signal is locked to its past by adjusting the DC bias voltage of the used Mach-Zehnder modulator. In this way, the oscillating frequency stability can be improved. Proving experiments on a 10-GHz OEO are performed. The results show that more than 20 dB improvements in the phase noise at low offset frequencies are achieved, the frequency fluctuation is reduced to less than 4 kHz in an hour, and the corresponding Allan deviation is decreased by 1 order of magnitude owing to the SPLL. This frequency stabilization scheme without use of any external reference sources has a great prospect in an integrated OEO.

Phase-coded microwave signal generation with a built-in optoelectronic oscillator based on a dual-parallel Mach-Zehnder modulator.

A scheme for binary phase-coded microwave signal generation is proposed and experimentally demonstrated with a dual-parallel Mach-Zehnder modulator (DPMZM). The carrier of the generated phase-coded signal is provided by the built-in dual-loop optoelectronic oscillator (OEO) based on the DPMZM. The principle of this signal generator and the oscillating condition of the OEO is theoretically described in detail. It is found that phase-coded signals can be produced by setting the three working points and the modulation depth of the DPMZM. In the proving experiment, a 10-GHz oscillating signal with a side mode suppression ratio of 49 dB is generated. The phase noise of this carrier wave is measured to be -80dBc/Hz at 10 kHz offset with a 100-m and 1-km optical fiber used in the two oscillation loops. When 1-Gbit/s and 2-Gbit/s coding signals are, respectively, applied to the DPMZM, two 10-GHz self-oscillating phase-coded signals are eventually produced, and the π phase shift in the signals is successfully confirmed with the phase information extracted from the phase-coded signals.

Natural soluble epoxide hydrolase inhibitors from Inula britanica and their potential interactions with soluble epoxide hydrolase: Insight from inhibition kinetics and molecular dynamics.

Soluble epoxide hydrolase (sEH) is a potential drug target to treat inflammation and neurodegenerative diseases. In this study, we found that the extract of Inula britanica exhibited significantly inhibitory effects against sEH, therefore, we investigated its phytochemical constituents to obtain seven new compounds together with sixteen known ones (1-20), including two pairs of novel enantiomers, (2S,3S)-britanicafanin A (1a), (2R,3R)-britanicafanin A (1b), (2R,3S)-britanicafanin B (2a), and (2S,3R)-britanicafanin B (2b), and three new lignans britanicafanins C-E (3-5). Their structures were determined by HRESIMS, 1D and 2D NMR, and electronic circular dichroism (ECD) spectra as well as quantum chemical computations. All the isolates were evaluated for their inhibitory effects against sEH, compounds 1-3, 5-7, 9, 10, 13, 14, and 17-20 showed significant inhibitory effects against sEH with IC50 values from 3.56 µM to 26.93 µM. The inhibition kinetics results indicated that compounds 9, 10, 13, and 19 were all uncompetitive inhibitors, and their inhibition constants (Ki) values were 7.11, 1.99, 4.06, and 8.78 µM, respectively. Their potential interactions were analyzed by molecular docking and molecular dynamics (MD), which suggested that amino acid residues Asp335 and Asn359, especially Gln384, played an important role in the inhibition of compounds 10 and 13 on sEH, and compounds 10 and 13 could be considered as the potential candidates for the development of sEH inhibitors.

Establishment of a homogeneous immunoassay-light-initiated chemiluminescence assay for detecting anti-Müllerian hormone in human serum.

Anti-Müllerian hormone (AMH) is known as a reliable marker of ovarian reserve (OR). The determination of AMH is of great importance and most existed AMH detection methods are heterogeneous immunoassay. In this study, a novel homogeneous sandwich immunoassay-light-initiated chemiluminescence assay (LICA) for detecting AMH serum level was developed. This AMH-LICA was performed by incubating serum samples with AMH mouse monoclonal antibody coated with chemibeads, streptavidin-coated sensibeads, and biotinylated AMH mouse monoclonal antibody. Sensitivity, precision, accuracy and cross-reactivity of this assay were evaluated. Besides, a regression analysis showed a high correlation between AMH-LICA and Roche Elecsys® AMH assay (y = 0.9851x + 0.07147, R2 = 0.9569). As a homogeneous immunoassay, this AMH-LICA could accurately and rapidly determine the serum level of AMH with high-throughput. Thus, this new developed assay may be a new useful analytical tool for the determination of AMH.

The clinical course of critically ill COVID-19 patients receiving invasive mechanical ventilation with subsequent terminal weaning: Primary data from 11 cases.

ABSTRACT: The coronavirus disease (COVID-19) outbreak was first reported in December 2019 in Wuhan, China. Specific information about critically ill COVID-19 patients receiving invasive mechanical ventilation (IMV) is rare.To describe the clinical course and complications of critically ill patients with COVID-19 who received IMV and were successfully weaned from it.This retrospective study included patients admitted to 3 intensive care units (ICUs) and 1 sub-ICU of Renmin Hospital of Wuhan University and Wuhan Jin Yin-tan Hospital between December 24, 2019, and March 12, 2020. Eleven patients who had been diagnosed with critically ill COVID-19 according to the World Health Organization interim guidance, received invasive ventilation, and were finally successfully weaned from it, were enrolled in our study. Their presenting symptoms, comorbidity conditions, laboratory values, ICU course, ventilator parameters, treatments, and relative complications were recorded.Of 108 critically ill COVID-19 patients who received invasive ventilation, 11 patients who underwent tracheal extubation or terminal weaning were included. The mean age of the 11 patients was 52.8 years (range, 38-70 years), 8 (72.7%) were male, and 2 were health care workers. The median time from onset of symptoms to dyspnea was 6.6 days (range, 3-13 days), and the median duration of IMV was 15.7 days (range, 6-29 days). All 11 patients presented with acute severe hypoxemic respiratory failure and received IMV, and 1 patient switched to extracorporeal membrane oxygenation assistance. A lung-protective strategy with lower tidal volume ventilation and proper driving pressure is the main strategy of IMV. All patients had extrapulmonary manifestations, including acute kidney injury, hepatic dysfunction, myocardial damage, and/or lymphopenia. Hospital-acquired infections occurred in 7 (63.6%) patients.Critical COVID-19 illness is characterized by acute hypoxemic respiratory failure and subsequent dysfunction of other organs with a high mortality rate. Correct ventilation strategies and other clinical strategies to improve oxygenation based on the skilled trained group and the availability of equipment are the key methods to rescue lives.

Recent advances in chemistry and bioactivity of Sargentodoxa cuneata.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Sargentodoxa comprises only one species, Sargentodoxa cuneata (Oliv.) Rehd et al., widely distributed in the subtropical zone of China. The plant is extensively used in traditional medicine for treating arthritis, joint pains, amenorrhea, acute appendicitis and inflammatory intestinal obstruction. Pharmacological studies show anti-inflammatory, antioxidant, antitumor, antimicrobial, and anti-sepsis activities. AIM OF THE REVIEW: This review aims to summarize the information about distribution, traditional uses, chemical constituents and pharmacological activities of S. cuneata, as an attempt to provide a scientific basis for its traditional uses and to support its application and development for new drug development. METHODOLOGY: Scientific information of S. cuneata was retrieved from the online bibliographic databases, including Web of Science, Google Scholar, PubMed, Springer Link, the Wiley online library, SciFinder, Baidu Scholar, China national knowledge infrastructure (CNKI) and WANFANG DATA (up to March 2020). We also search doctoral dissertations, master dissertations conference papers and published books. The keywords were used: "Sargentodoxa", "Da Xue Teng", "Hong Teng", "Xue Teng", "secondary metabolites", "chemical components", "biological activity", "pharmacology", "traditional uses". OBSERVATIONS AND RESULTS: S. cuneata is utilized as valuable herbal medicines to treat various diseases in China. Over 110 chemical constituents have been isolated and identified from the stem of S. cuneata, including phenolic acids, phenolic glycosides, lignans, flavones, triterpenoids and other compounds. The extract and compounds of S. cuneata have a wide spectrum of pharmacological activities, including antitumor, anti-inflammatory, antioxidant, antimicrobial, anti-sepsis and anti-arthritis effects, as well as protective activity against cerebrovascular diseases. CONCLUSION: S. cuneata has a rich legacy for the treatment of many diseases, especially arthritis and sepsis, which is reinforced by current investigations. However, the present studies about bioactive chemical constituents and detail pharmacological mechanisms of S. cuneata were insufficient. Further studies should focus on these aspects in relation to its clinical applications. This review has systematically summarized the traditional uses, phytochemical constituents and pharmacological effects of S. cuneata, providing references for the therapeutic potential of new drug development.

Oxidative stress in liver of turtle Mauremys reevesii caused by cadmium.

The research was designed to examine oxidative stress of the liver of turtle Mauremys reevesii caused by cadmium (Cd). Turtles were injected intraperitoneally with cadmium at the concentration of 7.5, 15, and 30 mg/kg, and 5 turtles were taken from each group after exposure for 1 week (1 w), 2 weeks (2 w), and 3 weeks (3 w). The activities of SOD and CAT as well as the contents of GSH and MDA in liver tissues were detected by using a kit. The results showed that the difference between the control group and the Cd-treated group was statistically significant with the increase of Cd concentration and the prolongation of exposure time, which suggested that Cd caused oxidative stress on the liver of turtles.

Lipid peroxidation of kidney of the turtle Mauremys reevesii caused by cadmium.

This research was designed to investigate lipid peroxidation of the kidney of turtle (Mauremys reevesii) caused by cadmium. Turtles were injected intraperitoneally with cadmium at the concentration of 0 (control), 7.5, 15, and 30 mg/kg, and 5 turtles were taken from each group after exposure for 1 week (1 w), 2 weeks (2 w), and 3 weeks (3 w). Superoxide dismutase (SOD) and catalase (CAT) activities as well as glutathione (GSH) and malonyldialdehyde (MDA) contents in the homogenate of kidney tissue were analyzed. The results demonstrated that a short time of low dose of cadmium could stimulate the increase of SOD activity in the kidney of turtles, but a long time of high dose of cadmium could induce the decrease of SOD activity in the kidney of turtles. Cadmium could decrease CAT activity and GSH content in turtle kidney, but increased MDA content in turtle kidney. There were some other effects on the turtles, such as depression and diarrhea. The experimental results indicate that cadmium causes temporary oxidative stress on the kidney of turtles.

Prognostic value of serum amyloid A in patients with COVID-19.

OBJECTIVE: To investigate the prognostic value of serum amyloid A (SAA) in the patients with Corona Virus Disease 2019 (COVID-19). METHODS: The medical data of 89 COVID-19 patients admitted to Renmin Hospital of Wuhan University from January 3, 2020 to February 26, 2020 were collected. Eighty-nine cases were divided into survival group (53 cases) and non-survival group (36 cases) according to the results of 28-day follow-up. The SAA levels of all patients were recorded and compared on 1 day after admission (before treatment) and 3 days, 5 days, and 7 days after treatment. The ROC curve was drawn to analyze the prognosis of patients with COVID-19 by SAA. RESULTS: The difference of comparison of SAA between survival group and non-survival group before treatment was not statistically significant, Z1 = - 1.426, P = 0.154. The Z1 values (Z1 is the Z value of the rank sum test) of the two groups of patients at 3 days, 5 days, and 7 days after treatment were - 5.569, - 6.967, and - 7.542, respectively. The P values were all less than 0.001, and the difference was statistically significant. The ROC curve results showed that SAA has higher sensitivity to the prognostic value of 1 day (before treatment), 3 days, 5 days, and 7 days after treatment, with values of 0.806, 0.972, 0.861, and 0.961, respectively. Compared with SAA on the 7th day and C-reactive protein, leukocyte count, neutrophil count, lymphocyte count, and hemoglobin on the 7th day, the sensitivities were: 96.1%, 83.3%, 88.3%, 83.3%, 67.9%, and 83.0%, respectively, of which SAA has the highest sensitivity. CONCLUSION: SAA can be used as a predictor of the prognosis in patients with COVID-19.

Natural sesquiterpenoid oligomers: A chemical perspective.

Sesquiterpenoid oligomers, biogenetically assembled by at least two monomeric sesquiterpenoid units via diverse pathways, represent a unique class of natural products with distinct bioactivities. Herein, we provide a review covering the dimeric, trimeric, and tetrameric sesquiterpenoids categorized by reaction types in biosynthesis from a chemical perspective. Emphasis is focused on the biosynthetic oligomerization pathways of these interesting molecules and their related biological functions, which will supply inspiration for the total synthesis or biomimetic synthesis of more oligomeric sesquiterpenoids and further pharmacological investigations.