Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Avidity Maturation and Association with Disease Severity.

The kinetics of IgG avidity maturation during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was studied. The IgG avidity assay, using a novel label-free immunoassay technology, revealed a strong correlation between IgG avidity and days since symptom onset. Peak readings were significantly higher in severe than mild disease cases.

A SARS-CoV-2 Label-Free Surrogate Virus Neutralization Test and a Longitudinal Study of Antibody Characteristics in COVID-19 Patients.

Methods designed to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humoral response include virus neutralization tests to determine antibody neutralization activity. For ease of use and universal applicability, surrogate virus neutralization tests (sVNTs) based on antibody-mediated blockage of molecular interactions have been proposed. A surrogate virus neutralization test was established on a label-free immunoassay platform (LF-sVNT). The LF-sVNT analyzes the binding ability of SARS-CoV-2 spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) after neutralizing RBD with antibodies in serum. The LF-sVNT neutralizing antibody titers (50% inhibitory concentration [IC50]) were determined from serum samples (n = 246) from coronavirus disease 2019 (COVID-19) patients (n = 113), as well as the IgG concentrations and the IgG avidity indices. Although there was variability in the kinetics of the IgG concentrations and neutralizing antibody titers between individuals, there was an initial rise, plateau, and then in some cases a gradual decline at later time points after 40 days after symptom onset. The IgG avidity indices, in the same cases, plateaued after an initial rise and did not show a decline. The LF-sVNT can be a valuable tool in research and clinical laboratories for the assessment of the presence of neutralizing antibodies to COVID-19. This study is the first to provide longitudinal neutralizing antibody titers beyond 200 days post-symptom onset. Despite the decline of IgG concentration and neutralizing antibody titer, IgG avidity index increases, reaches a plateau, and then remains constant up to 8 months postinfection. The decline of antibody neutralization activity can be attributed to the reduction in antibody quantity rather than the deterioration of antibody quality, as measured by antibody avidity.

Development of Label-Free Immunoassays as Novel Solutions for the Measurement of Monoclonal Antibody Drugs and Antidrug Antibodies.

BACKGROUND: Immunoassays based on label-free technologies (label-free immunoassay [LFIA]) offer an innovative approach to clinical diagnostics and demonstrate great promise for therapeutic drug monitoring (TDM) of monoclonal antibody (mAb) drugs. An LFIA measures immunocomplex formation in real time and allows for quantification on initial binding rate, which facilitates fast measurement within a few minutes. METHODS: Based on thin-film interferometry (TFI) technology, open-access LFIAs were developed for the quantification of the mAb drugs adalimumab (ADL) and infliximab (IFX) and for the detection of the antidrug antibodies (ADAs) to the mAb drugs (ADL-ADAs and IFX-ADAs). RESULTS: The LFIAs for active mAb drugs (ADL and IFX) and for ADAs (ADL-ADAs and IFX-ADAs) were validated. The analytical measurement range (AMR) for both ADL and IFX was from 2 to 100 µg/mL. The AMR for ADL-ADAs was from 5 to 100 µg/mL and for IFX-ADAs was 10 to 100 µg/mL. In the comparison of LFIAs and reporter gene assays, the correlation coefficient was 0.972 for the quantification of ADL and 0.940 for the quantification of IFX. The concordance rate was 90% for the detection of ADL-ADAs and 76% for the detection of IFX-ADAs. CONCLUSIONS: The LFIAs for active mAb drugs and ADAs were appropriate for the TDM of ADL and IFX. The TFI technology has unique advantages compared with other technologies used for the measurement of mAb drugs. Label-free technologies, especially those allowing for open-access LFIAs, have great potential for clinical diagnostics.

Nanoparticle Mobility over a Surface as a Probe for Weak Transient Disordered Peptide-Peptide Interactions.

Weak interactions form the core basis of a vast number of biological processes, in particular, those involving intrinsically disordered proteins. Here, we establish a new technique capable of probing these weak interactions between synthetic unfolded polypeptides using a convenient yet efficient, quantitative method based on single particle tracking of peptide-coated gold nanoparticles over peptide-coated surfaces. We demonstrate that our technique is sensitive enough to observe the influence of a single amino acid mutation on the transient peptide-peptide interactions. Furthermore, the effects of buffer salinity, which are expected to alter weak electrostatic interactions, are also readily detected and examined in detail. The method presented here has the potential to evaluate, in a high-throughput manner, weak interactions for a wide range of disordered proteins, polypeptides, and other biomolecules.

Colloid supported lipid bilayers for self-assembly.

The use of colloid supported lipid bilayers (CSLBs) has recently been extended to create colloidal joints, that enable the assembly of structures with internal degrees of flexibility, and to study lipid membranes on curved and closed geometries. These novel applications of CSLBs rely on previously unappreciated properties: the simultaneous fluidity of the bilayer, lateral mobility of inserted (linker) molecules and colloidal stability. Here we characterize every step in the manufacturing of CSLBs in view of these requirements using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Specifically, we have studied the influence of different particle properties (roughness, surface charge, chemical composition, polymer coating) on the quality and mobility of the supported bilayer. We find that the insertion of lipopolymers in the bilayer can affect its homogeneity and fluidity. We improve the colloidal stability by inserting lipopolymers or double-stranded inert DNA into the bilayer. We include surface-mobile DNA linkers and use FRAP to characterize their lateral mobility both in their freely diffusive and bonded state. Finally, we demonstrate the self-assembly of flexibly linked structures from the CSLBs modified with surface-mobile DNA linkers. Our work offers a collection of experimental tools for working with CSLBs in applications ranging from controlled bottom-up self-assembly to model membrane studies.

Glucose-based biofuel cells and their applications in medical implants: A review.

In glucose biofuel cells (G-BFCs), glucose oxidation at the anode and oxygen reduction at the cathode yield electrons, which generate electric energy that can power a wide range of electronic devices. Research associated with the development of G-BFCs has increased in popularity among researchers because of the eco-friendly nature of G-BFCs (as related to their construction) and their evolution from inexpensive bio-based materials. In addition, their excellent specificity towards glucose as an energy source, and other properties, such as small size and weight, make them attractive within various demanding applied environments. For example, G-BFCs have received much attention as implanted devices, especially for uses related to cardiac activities. Envisioned pacemakers and defibrillators powered by G-BFCs would not be required to have conventional lithium batteries exchanged every 5-10 years. However, future research is needed to develop G-BFCs demonstrating more stable power consistency and improved lifespan, as well as solving the challenges in converting laboratory-made implantable G-BFCs into implanted devices in the human body. The categorization of G-BFCs as a subcategory of different biofuel cells and their performance is reviewed in this article.

Shear-directed assembly of graphene oxide in aqueous dispersions into ordered arrays.

A wide variety of new carbon-based materials are being developed from graphene oxide (GO) precursor sheets, whose assembly in aqueous phases determines the form, structure, and properties of the resultant carbon. Here we show that graphene oxide forms ordered linear arrays of aggregates when aqueous suspensions are subjected to shear flow in the presence of soluble salts. These linear arrays align along the vorticity direction, normal to the direction of flow. We propose that salt addition screens electrostatic repulsion and allows formation of fractal-like GO sheet aggregates by hydrophobic forces. Fluid shear in a confined gap then guides the assembly of these primary aggregates into optically visible, ordered linear arrays or "superaggregates" whose characteristics are a function of GO concentration, salt valency, salt concentration, and gap confinement. This is the first reported observation of vorticity banding in graphene oxide suspensions and the first reported observation of such banding based on salt-induced interactions. We also demonstrate that simple isometric nanoparticles of carbon or gold do not form such linear superaggregate arrays but can be assembled into such arrays using graphene oxide as a two-dimensional colloidal template.

Brownian motion of flexibly linked colloidal rings.

Ring, or cyclic, polymers have unique properties compared to linear polymers, due to their topologically closed structure that has no beginning or end. Experimental measurements on the conformation and diffusion of molecular ring polymers simultaneously are challenging due to their inherently small size. Here, we study an experimental model system for cyclic polymers, that consists of rings of flexibly linked micron-sized colloids with n=4-8 segments. We characterize the conformations of these flexible colloidal rings and find that they are freely jointed up to steric restrictions. We measure their diffusive behavior and compare it to hydrodynamic simulations. Interestingly, flexible colloidal rings have a larger translational and rotational diffusion coefficient compared to colloidal chains. In contrast to chains, their internal deformation mode shows slower fluctuations for n≲8 and saturates for higher values of n. We show that constraints stemming from the ring structure cause this decrease in flexibility for small n and infer the expected scaling of the flexibility as function of ring size. Our findings could have implications for the behavior of both synthetic and biological ring polymers, as well as for the dynamic modes of floppy colloidal materials.

Controlled clustering in metal nanorod arrays leads to strongly enhanced field emission characteristics.

We show that controlled clustering in electrochemically grown silver nanorods results in up to 50% enhancement in their field emission performance. Larger cluster size and nanorod length lead to a lower turn-on electric field, a higher current density and a larger enhancement factor. However, beyond a critical length (≈30 µm), the nanorods begin to form disordered ridges instead of well separated conical clusters and the field emission performance proceeds to deteriorate. With the help of finite element modelling, we show that a larger cluster size indeed leads to an enhanced electric field at the cluster edges, effectively reducing screening effects and thereby enhancing the field emission performance. Though our present work pertains to silver nanorods, a similar clustering of nanorods is observed in many types of electrochemically grown nanorods, and even in carbon nanotubes and Si nanowires grown by other techniques. Hence, we expect this study to have general applicability in the design of better nanorod-based field emitters.

Self-Assembly Dynamics of Reconfigurable Colloidal Molecules.

Colloidal molecules are designed to mimic their molecular analogues through their anisotropic shape and interactions. However, current experimental realizations are missing the structural flexibility present in real molecules thereby restricting their use as model systems. We overcome this limitation by assembling reconfigurable colloidal molecules from silica particles functionalized with mobile DNA linkers in high yields. We achieve this by steering the self-assembly pathway toward the formation of finite-sized clusters by employing high number ratios of particles functionalized with complementary DNA strands. The size ratio of the two species of particles provides control over the overall cluster size, i.e., the number of bound particles N, as well as the degree of reconfigurability. The bond flexibility provided by the mobile linkers allows the successful assembly of colloidal clusters with the geometrically expected maximum number of bound particles and shape. We quantitatively examine the self-assembly dynamics of these flexible colloidal molecules by a combination of experiments, agent-based simulations, and an analytical model. Our "flexible colloidal molecules" are exciting building blocks for investigating and exploiting the self-assembly of complex hierarchical structures, photonic crystals, and colloidal metamaterials.

A thin-film interferometry-based label-free immunoassay for the detection of daratumumab interference in serum protein electrophoresis.

BACKGROUND: Daratumumab (DARA) is a fully human anti-CD38 IgG1-κ monoclonal antibody drug used in the treatment of multiple myeloma (MM). While serum protein electrophoresis (SPEP) is an important assay for diagnosis and monitoring of patients with MM, DARA can appear in the γ-region as a single band and interfere with the interpretation of SPEP results. An approach to detect the interference is measuring the quantity of DARA in serum samples and assessing its impact on SPEP results. Immunoassays based on label-free technologies, i.e. label-free immunoassays (LFIA's), can achieve real-time immunometric measurement without attaching a reporter molecule (enzyme, fluorophore, etc.) to the immunocomplex. The recorded time course of the immunocomplex formation allows for quantitation on initial binding rate, which facilitates rapid measurement within a few minutes. Based on the thin-film interferometry (TFI) technology, a rapid LFIA was established for the quantitation of DARA in serum samples. METHODS: The TFI-based LFIA for DARA was validated for imprecision (CV), accuracy, limit of quantitation (LOQ), and analytical measurement range (AMR). Interference to the LFIA was evaluated using a group of protein samples, as well as hemolytic, lipemic, and icteric clinical samples. RESULTS: The precision of the TFI-based LFIA's for DARA ranged from 6.5% to 10.7% (within-run CV), and 7.4% to 11.6% (between-run CV), with a bias of -2.1% to 10.1%. The LOQ was 10 µg/ml (n = 4, CV 9.8%), with an AMR ranging from the LOQ to 1000 µg/ml. The LFIA was used to measure 37 patient samples submitted for SPEP testing. The LFIA results were 100% consistent with the history of DARA use as documented in the medical record. CONCLUSIONS: The TFI-based LFIA was successful at accurately identifying DARA in serum samples and can be used to identify DARA interference in SPEP testing. This work demonstrates the applicability of label-free technologies, particularly the TFI technology, to clinical diagnostic needs. Given the simplicity and the speed of the testing process, the TFI technology provides a unique testing approach for the measurement of proteins in clinical samples.

Attenuation of haemodynamic responses to laryngoscopy and endotracheal intubation with dexmedetomidine: A comparison between intravenous and intranasal route.

BACKGROUND AND AIMS: Haemodynamic changes during endotracheal intubation are major concerns in general anaesthesia This study compared the efficacy of intranasal and intravenous dexmedetomidine (DEX) to attenuate the stress response of laryngoscopy and endotracheal intubation. METHODS: In this prospective, randomised, double-blinded study, 70 adults were divided into two groups [Group DIV(n=35) and Group DIN(n=35)]. DIV group received intravenous dexmedetomidine (DEX) infusion (0.5 µg/kg) over 40 min and DIN group received intranasal dexmedetomidine (1 µg/kg) 40 min before induction. The primary objective was the comparison the mean arterial pressure (MAP) between two groups from 40 min before induction at every 10 min intervals till induction of anaesthesia, at the time of intubation, thereafter every 1 min interval till 5 min, at 7 min and 10 min after intubation. The secondary outcomes were comparison of heart rate, systolic and diastolic blood pressure along with sedation and other adverse effects. Statistical analysis was with Statistica 6.0 and Graph Pad prism version 5. RESULTS: In both the groups, all the haemodynamic parameters were maintained within (20% of baseline values) throughout the study period. There was no statistically significant difference in MAP between two groups (P>0.05). Preoperative sedation score was significantly higher in the DIV group than the DIN group (P = 0.014). CONCLUSION: Like IV DEX, intranasal DEX can also attenuate the haemodynamic stress responses of laryngoscopy and endotracheal intubation without significant differences in MAP between two groups.

Pyrimidine-based inhibitors of CaMKIIdelta.

Non-ATP competitive pyrimidine-based inhibitors of CaMKIIdelta were identified. Computational studies were enlisted to predict the probable mode of binding. The results of the computational studies led to the design of ATP competitive inhibitors with optimized hinge interactions. Inhibitors of this class possessed improved enzyme and cellular activity compared to early leads.

Efficacy of intravenous dexmedetomidine on patient's satisfaction, comfort and sedation during awake fibre-optic intubation in patients with cervical spondylotic myelopathy posted for elective cervical fixation.

BACKGROUND AND AIMS: Various anaesthetic drugs, in addition to airway block, are used for producing favourable intubation conditions during awake fibre-optic intubation (AFOI), but most of them cause respiratory depression and hypoxaemia. The aim of this study was to evaluate the efficacy of intravenous (IV) dexmedetomidine (DEX) on sedation, patient comfort and cardiovascular responses during AFOI in patients with cervical spondylotic myelopathy (CSM). METHODS: This randomised, placebo-controlled, double-blinded, prospective study was conducted on 56 adult patients with cervical spondylotic myelopathy (CSM) undergoing elective cervical fixation, who were randomly allocated into two groups - Group D and Group C. Group D patients received DEX infusion at a rate of 1 µg/kg for the first 10 min followed by 0.5 µg/kg/h and Group C received 0.9% normal saline infusion in the same manner. Airway blocks with lignocaine were given to all patients before undergoing AFOI. Patient's alertness, sedation and cardiorespiratory changes during the procedure were assessed by the Observer Assessment Awareness and Sedation (OAA/S) scale. On the 1st post-operative day, patient's' comfort during AFOI was assessed by visual analogue scale (VAS). RESULTS: Patients of Group D had an acceptable level of sedation (OAA/S score: 20 to 17 with greater comfort and satisfaction (VAS: 40-60), compared to control group (VAS: 50-90, P < 0.001.). Moreover, haemodynamic parameters were less significantly altered in the DEX group during AFOI. CONCLUSIONS: IV DEX infusion during AFOI improves patient's tolerances with an acceptable level of sedation without significant haemodynamic instability and respiratory depression.

Colloidal joints with designed motion range and tunable joint flexibility.

The miniaturization of machines towards the micron and nanoscale requires the development of joint-like elements that enable and constrain motion. We present a facile method to create colloidal joints, that is, anisotropic colloidal particles functionalized with surface mobile DNA linkers that control the motion range of bonded particles. We demonstrate quantitatively that we can control the flexibility of these colloidal joints by tuning the DNA linker concentration in the bond area. We show that the shape of the colloidal joint controls the range of motion of bonded particles through a maximisation of the bond area. Using spheres, cubes, and dumbbells, we experimentally realize spherical joints, planar sliders, and hinges, respectively. Finally we demonstrate the potential of the colloidal joints for programmable bottom-up self-assembly by creating flexible colloidal molecules and colloidal polymers. The reconfigurability and motion constraint offered by our colloidal joints make them promising building blocks for the development of switchable materials and nanorobots.

Efficacy of transdermal buprenorphine patch on post-operative pain relief after elective spinal instrumentation surgery.

BACKGROUND AND AIMS: Transdermal buprenorphine patch (TDB) is increasingly used for chronic pain management because of non-invasive dosing, longer duration of action and minimal side effects. However its role in acute post-operative pain management for spinal instrumentation surgery is not well established. The aim of this study was to evaluate the analgesic efficacy of buprenorphine patch for postoperative pain relief in patients undergoing spinal instrumentation surgery. METHODS: In this randomised, placebo-controlled, double-blinded, prospective study, 70 adult patients undergoing elective spinal instrumentation surgery were randomly allocated into two groups-TDB Group (buprenorphinepatch) and TDP Group (placebo patch). Time to first rescue analgesic requirement was the primary outcome. All patients also were monitored for total rescue analgesic requirement, drug-related adverse effect and haemodynamic status till 48 h after surgery. Statistical analysis was carried out using student independent t-test if normally distributed or with Mann-Whitney U-test if otherwise. RESULTS: Time to first post-operative rescue analgesic (tramadol) requirement was much delayed in TDB Group than TDP Group (708.0 ± 6.98 min vs 54 ± 0.68 min, P < 0.001) and the total tramadol requirement was higher in TDB Group (490.60 ± 63.09 averagevs. 162.93 ± 63.91 mg, P < 0.001). Intra-and post-operative haemodynamic status was also stable in TDB Group without any adverse event. CONCLUSION: A TDB patch (10 µg/hour) applied 24 hours before surgery can be used as a postoperative analgesic for lumber fixation surgery without any drug-related adverse effect.

Efficacy and safety of empagliflozin in type 2 diabetes mellitus: a meta-analysis of randomized controlled trials.

OBJECTIVES: The prevalence of diabetes has increased in the recent decades and optimum glycemic control is required to reduce morbidity and mortality. We meta-analyzed randomized controlled trials in order to assess the efficacy and safety of empagliflozin compared to placebo in type 2 diabetes mellitus patients. METHODS: We included double-blind, placebo controlled trials of empagliflozin that evaluated glycemic efficacy and safety (10 mg or 25 mg) either as monotherapy or as add-on to existing diabetes pharmacotherapy. RESULTS: The results demonstrated significant improvements in HbA1c (SMD -0.929%, 95 % CI -1.064 to -0.793, for 10 mg and -1.064%, 95 % CI -1.184 to -0.944, for 25 mg) and FPG (SMD -0.929%, 95 % CI -1.064 to -0.793, for 10 mg and -1.064%, 95 % CI -1.184 to -0.944, for 25 mg) with empagliflozin monotherapy (n = 609) compared to placebo. Significant improvements in HbA1c [SMD -1.582%, 95% CI -2.164 to -1.000, for 10 mg (n = 1079) and -1.668%, 95% CI -2.260 to -1.077, for 25 mg (n = 1070)] and FPG [SMD -0.865 mmol/L, 95 % CI -1.309 to -0.420, for 10 mg (n = 854) and -0.996 mmol/L, 95% CI -1.456 to -0.536, for 25 mg (n = 854)] were also observed in empagliflozin add-on therapy trials. Reductions in blood pressure and body weight were also seen in both monotherapy and add-on therapy. Empagliflozin was associated with increased risk of hypoglycemia, genital and urinary tract infections (OR 1.043, 2.814, 1.119 respectively). CONCLUSION: This meta-analysis shows empagliflozin is safe and effective for the treatment of T2DM along with existing diabetes pharmacotherapy.

Effects of ultra high dilution of paraquat 30 ch on growth, chlorophyll content and yield of rice crop

The application of synthetic fertilizers reduces the natural fertility of the soil and contaminates groundwater. Some photosynthesis inhibitors at ultra-high dilution (UHD) increase photosynthesis, growth, and yield of crops. A weedicide Paraquat at UHD enhanced the growth and yield of potatoes in fields. The objective is to see whether the UHD of Paraquat is also effective on rice. This weedicide was serially diluted with distilled water and manually succussed in 30 steps following the preparation of homeopathic dilutions called potencies. In this way, the 30thpotency of Paraquat called Paraquat 30 cH was prepared and preserved in 90 % ethanol. Paraquat 30 cH was diluted with water 1:1000 (v/v) and sprayed on rice plants in a field measuring 0.3125 acres. The control plot of the same area was situated 300 meters away from the test plot. Three treatments were given at an interval of 7 days. The treated plot showed increased growth, chlorophyll content, and rice yield significantlycompared to control. The UHD of the weedicide produced precisely the opposite effect of the crude material on plants. The increased growth and yield of rice by Paraquat 30 cH may be due to the enhancement of photosynthesis of treated plants. The UHD of Paraquat increased the yield of rice by 19.35% over the control.

Extreme dilutions of mercuric chloride produce longer binding interaction with ?-amylase as compared to blank solvents

Background:High and ultra-low doses of HgCl2modulate α-amylase starch interaction through two different binding sites of the enzyme. Mercurius corrosivus at ultra-high dilution (UHD) is reported to have a long duration of action for 30-60 days.Objectives:(i) To see whether the mother tincture and UHDs of mercuric chloride could directly act on α-amylase without intervention of its substrate starch. (ii) To see whether the MT and UHDs produce longer binding interaction with α-amylase than the control.Methods:Mercuric chloride MT (0.15 M) or Merc corθ and its three UHDs, 30, 200, 1000 cH called potencies were in 90% ethanol. Blank 90% ethanol and deionised and distilled (DD) water were used as controls. All ethanol based ligands were diluted with DD water to reduce EtOH content to 0.09%. Using an ITC instrument each ligand was injected once at 10 µl into15µM α-amylase at 250C and the reaction was observed for 33.3 min. Results:While Merccorθ produced endothermic reaction, all other ligands did exothermic reaction. All ligands sustained heat change for the entire period of observation (33.3 min). Conclusions:Merc corθ and potencies produced exactly the opposite reaction at the protein binding sites. Merc cor200 cH and 1000 cH produced highest heat change (6 micro cal/sec) during interaction with α-amylase.(AU)

The nature of the structural phase transition from the hexagonal (4H) phase to the cubic (3C) phase of silver.

The phase transition from the hexagonal 4H polytype of silver to the commonly known 3C (fcc) phase was studied in detail using x-ray diffraction, electron microscopy, differential scanning calorimetry and Raman spectroscopy. The phase transition is irreversible and accompanied by extensive microstructural changes and grain growth. Detailed scanning and isothermal calorimetric analysis suggests that it is an autocatalytic transformation. Though the calorimetric data suggest an exothermic first-order phase transition with an onset at 155.6 °C (for a heating rate of 2 K min(-1)) and a latent heat of 312.9 J g(-1), the microstructure and the electrical resistance appear to change gradually from much lower temperatures. The 4H phase shows a Raman active mode at 64.3 cm(-1) (at 4 K) that undergoes mode softening as the 4H → 3C transformation temperature is approached. A first-principles density functional theory calculation shows that the stacking fault energy of 4H-Ag increases monotonically with temperature. That 4H-Ag has a higher density of stacking faults than 3C-Ag, implies the metastability of the former at higher temperatures. Energetically, the 4H phase is intermediate between the hexagonal 2H phase and the 3C ground state, as indicated by the spontaneous transformation of the 2H to the 4H phase at -4 °C. Our data appear to indicate that the 4H-Ag phase is stabilized at reduced dimensions and thermally induced grain growth is probably responsible for triggering the irreversible transformation to cubic Ag.