Screening of MnO2 with desired facet and its behavior in highly selective adsorption of aqueous Pb (II): Theoretical and experimental studies.

In this study, Density Functional Theory (DFT) calculations and experimental methods were adopted to evaluate MnO2 with 5 different facets for their selective adsorption of Pb (II) from wastewater containing Cd (II), Cu (II), Pb (II), and Zn (II). The DFT calculations were performed to screen the selective adsorption capability of the facets and demonstrated that the MnO2 (3 1 0) facet has an excellent performance in selective adsorption of Pb (II) among all facets. The validity of DFT calculations was verified by comparing with the experimental results. MnO2 with different facets was prepared in a controlled manner and the characterizations confirmed that the lattice indices of the fabricated MnO2 have the desired facets. Adsorption performance experiments illustrated a high adsorption capacity (320.0 mg/g) on the (3 1 0) facet MnO2. The selectivity of adsorption of Pb (II) was 3-32 times greater than that of the other coexisting ions, i.e., Cd (II), Cu (II), and Zn (II)), which is consistent with results of the DFT calculations. Furthermore, results of DFT calculations on adsorption energy, charge density difference, and projected density of states (PDOS) showed that the adsorption of Pb (II) on the MnO2 (3 1 0) facet is non-activated chemisorption. This study shows that it is feasible to use DFT calculations to quickly screen suitable adsorbents for environmental applications.

Recent Advances in Graphitic Carbon Nitride Based Electro-Catalysts for CO2 Reduction Reactions.

The electrocatalytic carbon dioxide reduction reaction is an effective means of combating the greenhouse effect caused by massive carbon dioxide emissions. Carbon nitride in the graphitic phase (g-C3N4) has excellent chemical stability and unique structural properties that allow it to be widely used in energy and materials fields. However, due to its relatively low electrical conductivity, to date, little effort has been made to summarize the application of g-C3N4 in the electrocatalytic reduction of CO2. This review focuses on the synthesis and functionalization of g-C3N4 and the recent advances of its application as a catalyst and a catalyst support in the electrocatalytic reduction of CO2. The modification of g-C3N4-based catalysts for enhanced CO2 reduction is critically reviewed. In addition, opportunities for future research on g-C3N4-based catalysts for electrocatalytic CO2 reduction are discussed.

Computational screening of effective g-C3N4 based single atom electrocatalysts for the selective conversion of CO2.

Two-dimensional (2D) material-based single-atom catalysts (SACs) have demonstrated their potential in electrochemical reduction reactions but exploring suitable 2D material-based SACs for the CO2 reduction reaction (CO2RR) by experiments is still a formidable task. In this study, theoretical screening of transition metal (TM)-doped graphitic carbon nitride (g-C3N4) materials as catalysts for the CO2RR was systematically performed based on density functional theory (DFT) calculations. An indicator for the selective formation of one carbon (C1) products was developed to screen catalysts that are active and selective in the CO2RR. The results indicated that Ti- and Ag-g-C3N4 demonstrate excellent catalytic activity and selectivity for the formation of CO and HCOOH, with limiting potentials of -0.330 and -0.096 V, respectively, while Cr-g-C3N4 exhibits the highest catalytic activity for yielding CH3OH and CH4 (-0.355 and -0.420 V, respectively), but none of the screened catalysts have been identified as ideal candidates for the selective production of CH3OH and CH4. Furthermore, Bader charge analysis suggested that excessive electron transfer from TM leads to stronger adsorption of intermediates and high limiting potentials, which subsequently result in lower catalytic activity. This work provides theoretical insights into the effective screening of active and selective 2D material-based SACs which has the potential to significantly reduce the time and resources required for the discovery of novel electrocatalysts for the controlled formation of various products.

Adsorptive removal of organic dyes via porous materials for wastewater treatment in recent decades: A review on species, mechanisms and perspectives.

Organic dyes, a type of high toxic and carcinogenic chemicals that present severe threats to human and aquatic life, are the most commonly seen organic pollutants in wastewater of industries such as textile, rubber, cosmetic industry etc. Various techniques for the removal of dyes are compared in this review. Adsorption has proven to be a facile and promising approach for the removal of dyes in wastewater. This work focuses on the latest development of various porous materials for the adsorption of organic dyes. The characteristics, functionalization and modification of different porous materials are also presented. Furthermore, adsorption behaviors and mechanism of these adsorbents in the adsorption of organic dyes are critically reviewed. Finally, challenges and opportunities for future research in the development of novel materials for the highly efficient removal of dyes are proposed.

Mitophagy in the basolateral amygdala mediates increased anxiety induced by aversive social experience.

Psychosocial stress is a common risk factor for anxiety disorders. The cellular mechanism for the anxiogenic effect of psychosocial stress is largely unclear. Here, we show that chronic social defeat (CSD) stress in mice causes mitochondrial impairment, which triggers the PINK1-Parkin mitophagy pathway selectively in the amygdala. This mitophagy elevation causes excessive mitochondrial elimination and consequent mitochondrial deficiency. Mitochondrial deficiency in the basolateral amygdalae (BLA) causes weakening of synaptic transmission in the BLA-BNST (bed nucleus of the stria terminalis) anxiolytic pathway and increased anxiety. The CSD-induced increase in anxiety-like behaviors is abolished in Pink1-/- and Park2-/- mice and alleviated by optogenetic activation of the BLA-BNST synapse. This study identifies an unsuspected role of mitophagy in psychogenetic-stress-induced anxiety elevation and reveals that mitochondrial deficiency is sufficient to increase anxiety and underlies the psychosocial-stress-induced anxiety increase. Mitochondria and mitophagy, therefore, can be potentially targeted to ameliorate anxiety.

Dysbindin-1 regulates mitochondrial fission and gamma oscillations.

Mitochondria are cellular ATP generators. They are dynamic structures undergoing fission and fusion. While much is known about the mitochondrial fission machinery, the mechanism of initiating fission and the significance of fission to neurophysiology are largely unclear. Gamma oscillations are synchronized neural activities that impose a great energy challenge to synapses. The cellular mechanism of fueling gamma oscillations has yet to be defined. Here, we show that dysbindin-1, a protein decreased in the brain of individuals with schizophrenia, is required for neural activity-induced fission by promoting Drp1 oligomerization. This process is engaged by gamma-frequency activities and in turn, supports gamma oscillations. Gamma oscillations and novel object recognition are impaired in dysbindin-1 null mice. These defects can be ameliorated by increasing mitochondrial fission. These findings identify a molecular mechanism for activity-induced mitochondrial fission, a role of mitochondrial fission in gamma oscillations, and mitochondrial fission as a potential target for improving cognitive functions.

The Projection From Ventral CA1, Not Prefrontal Cortex, to Nucleus Accumbens Core Mediates Recent Memory Retrieval of Cocaine-Conditioned Place Preference.

Drug-paired cues inducing memory retrieval by expressing drug-seeking behaviors present a major challenge to drug abstinence. How neural circuits coordinate for drug memory retrieval remains unclear. Here, we report that exposure of the training chamber where cocaine-conditioned place preference (CPP) was performed increased neuronal activity in the core of nucleus accumbens (AcbC), ventral CA1 (vCA1), and medial prefrontal cortex (mPFC), as shown by elevated pERK and c-Fos levels. Chemogenetic inhibition of neuronal activity in the vCA1 and AcbC, but not mPFC, reduced the time spent in the cocaine-paired compartment, suggesting that the vCA1 and AcbC are required for the retrieval of cocaine-CPP memory and are key nodes recruited for cocaine memory storage. Furthermore, chemogenetic inhibition of the AcbC-projecting vCA1 neurons, but not the AcbC-projecting mPFC neurons, decreased the expression of cocaine-CPP. Optogenetic inhibition of the vCA1-AcbC projection, but not the mPFC-AcbC projection, also reduced the preference for the cocaine-paired compartment. Taken together, the cue-induced natural recall of cocaine memory depends on vCA1-AcbC circuits. The connectivity from the vCA1 to the AcbC may store the information of the cue-cocaine reward association critically required for memory retrieval. These data thus provide insights into the neural circuit basis of retrieval of drug-related memory.

Autophagy controls the induction and developmental decline of NMDAR-LTD through endocytic recycling.

NMDA receptor-dependent long-term depression (NMDAR-LTD) is a long-lasting form of synaptic plasticity. Its expression is mediated by the removal of AMPA receptors from postsynaptic membranes. Under basal conditions, endocytosed AMPA receptors are rapidly recycled back to the plasma membrane. In NMDAR-LTD, however, they are diverted to late endosomes for degradation. The mechanism for this switch is largely unclear. Additionally, the inducibility of NMDAR-LTD is greatly reduced in adulthood. The underlying mechanism and physiological significance of this phenomenon are elusive. Here, we report that autophagy inhibition is essential for the induction and developmental dampening of NMDAR-LTD. Autophagy is inhibited during NMDAR-LTD to decrease endocytic recycling. Autophagy inhibition is both necessary and sufficient for LTD induction. In adulthood, autophagy is up-regulated to make LTD induction harder, thereby preventing the adverse effect of excessive LTD on memory consolidation. These findings reveal the unrecognized functions of autophagy in synaptic plasticity, endocytic recycling, and memory.

[Therapeutic effect on post-stroke spastic paralysis of upper extremity treated with combination of kinematic-acupuncture therapy and rehabilitation training].

OBJECTIVE: To compare the clinical therapeutic effect on post-stroke spastic paralysis of the upper extremity between the combination of kinematic-acupuncture therapy and rehabilitation training and the combined treatment of the conventional acupuncture with rehabilitation training. METHODS: A total of 60 patients of post-stroke spastic paralysis of the upper extremity at the non-acute stage were randomized into an observation group (30 cases) and a control group (30 cases, 1 case dropped off). On the base of the routine western medication and rehabilitation treatment, the kinematic-acupuncture therapy was added in the observation group and the conventional acupuncture was used in the control group. Baihui (GV 20), Dazhui (GV 14), Jiaji (EX-B 2) from T1 to T8, Tianzong (SI 11), Jianzhen (SI 9), Jianyu (LI 15) and Quyuan (SI 13) were selected in both groups. The treatment was given once daily and the treatment for 14 days was as one course. The one course of treatment was required in this research. Separately, before treatment and in 7 and 14 days of treatment, the score of simplified Fugl-Meyer scale of the upper extremity (FMA-UE), the grade of the modified Ashworth scale (MAS) and the score of the modified Barthel index scale (MBI) were compared between the two groups. RESULTS: Compared before treatment, in 7 and 14 days of treatment, FMA-UE score was increased obviously in either group (P<0.01). In 14 days of treatment, FMA-UE score in the observation group was higher than that in the control group (P<0.05). In 7 and 14 days of treatment, MAS grades of shoulder joint, elbow joint, wrist joint and metacarpophalangeal joint were all improved markedly in the two groups (P<0.05). Compared with the grades in 7 days of treatment, MAS grades of elbow joint and metacarpophalangeal joint were improved markedly in 14 days of treatment in the two groups (P<0.05). Compared with the control group, MAS grades of elbow joint and metacarpophalangeal joint were improved more markedly in the observation group in 14 days of treatment (P<0.05). Compared with the score before treatment, MBI score was increased in 7 and 14 days of treatment respectively in the observation group (P<0.05, P<0.01). In 14 days of treatment, MBI score was increased in the control group (P<0.01). CONCLUSION: For the patients with post-stroke spastic paralysis of the upper extremity at the non-acute stage, the combined treatment with kinematic-acupuncture therapy and rehabilitation training obviously improves the motor function of the upper extremity and the muscle tone of elbow joint and metacarpophalangeal joint. The therapeutic effect of this combination is better than that of the combined treatment of the conventional acupuncture with rehabilitation training. Additionally, this combined therapy improves the ability of daily life activity.

A ventral CA1 to nucleus accumbens core engram circuit mediates conditioned place preference for cocaine.

The importance of neuronal ensembles, termed engram cells, in storing and retrieving memory is increasingly being appreciated, but less is known about how these engram cells operate within neural circuits. Here we tagged engram cells in the ventral CA1 region of the hippocampus (vCA1) and the core of the nucleus accumbens (AcbC) during cocaine conditioned place preference (CPP) training and show that the vCA1 engram projects preferentially to the AcbC and that the engram circuit from the vCA1 to the AcbC mediates memory recall. Direct activation of the AcbC engram while suppressing the vCA1 engram is sufficient for cocaine CPP. The AcbC engram primarily consists of D1 medium spiny neurons, but not D2 medium spiny neurons. The preferential synaptic strengthening of the vCA1→AcbC engram circuit evoked by cocaine conditioning mediates the retrieval of cocaine CPP memory. Our data suggest that the vCA1 engram stores specific contextual information, while the AcbC D1 engram and its downstream network store both cocaine reward and associated contextual information, providing a potential mechanism by which cocaine CPP memory is stored.

[Mechanism of Synergistic Adsorption of Arsenic and Cadmium by Aluminium-substituted Ferrihydrites].

The synergistic process and mechanism of aluminum (Al)-substituted ferrihydrites on arsenic[As(Ⅴ)] and cadmium[Cd(Ⅱ)] were studied under laboratory conditions. The results showed that synergistic adsorption and coprecipitation of As and Cd by Al-substituted ferrihydrites was clearly affected by both the pH of solution and the order in which heavy metals were added. The solution in which As co-existed with Cd for 72 hours, at a pH of 6.0 to 6.5, the As and Cd adsorption capacity of Al-substituted ferrihydrites containing 20% Al (AF20) reached 60.9 mg·g-1 and 17.1 mg·g-1, respectively. The removal rates of As and Cd were 96.0% and 73.0%, respectively. Arsenic and Cd were synergistically adsorbed into the internal pores of AF20 particles, and the synergistic adsorption effect of AF20 on As and Cd was clear. Adding Cd to the solution containing As, for 72 hours, and with a pH of 6.1 to 6.5, the As and Cd adsorption capacity of AF20 was 58.1 mg·g-1 and 12.4 mg·g-1, respectively. The removal rates of As and Cd were 96.0% and 48.3%, respectively. Adsorption of As limited the fixation of Cd by AF20. When adding As to the solution containing Cd, for 72 hours, with a pH of 9.5 to 9.8, fixed amounts of As and Cd on AF20 were 20.9 mg·g-1 and 24.4 mg·g-1, respectively. The removal rates of As and Cd were 38.8% and 98.9%, respectively. The coprecipitation of As and Cd by AF20 was clear. The resulting insoluble As and Cd compounds distributed the Cd distribution in a sparse strip and impeded the further adsorption of As. The results show that Al-substituted ferrihydrites can synergistically adsorb and coprecipitate As and Cd in contaminated environmental media.

Release of cadmium in contaminated paddy soil amended with NPK fertilizer and lime under water management.

Agricultural soils contaminated with cadmium (Cd) pose a risk to receiving surface water via drainage or runoff. A 90-day laboratory incubation experiment was conducted to investigate the release characteristics and transformation of Cd from contaminated paddy soil amended with agrochemical (NPK fertilizer) and lime (L) under water management regimes of continuous flooding (F) and drying-wetting cycles (DW). The result showed that the dissolved Cd concentrations in overlying water of the fertilizer treatment under flooding (NPK+F) and drying-wetting (NPK+DW) reached up to 81.0 µg/L and 276 µg/L, and were much higher than that from the corresponding controls without NPK fertilizer addition at the end of experiment. The Cd concentration showed significantly negative correlation with overlying water pH, but positive correlation with soil redox potential and concentrations of dissolved total nitrogen, sulfate and manganese in overlying water (P < 0.05), indicating that drying-wetting cycles and N fertilizer addition may enhance soil Cd release. The Cd concentrations in overlying water from all treatments except NPK+L+F treatment exceeded the Cd threshold limit of Chinese Environmental Quality Standards for Surface Water (10 µg/L Grade V) and poses potential risk to surface water quality. Meanwhile, the proportion of Cd in the acid-soluble fraction from all incubated soil except NPK+L+F treatment increased compared to before incubation. The results indicated that continuous flooding was a reasonable water management candidate coupled with lime addition for immobilizing soil Cd.

ß1-Adrenoceptor in the Central Amygdala Is Required for Unconditioned Stimulus-Induced Drug Memory Reconsolidation.

Background: Drug memories become labile and reconsolidated after retrieval by presentation of environmental cues (conditioned stimulus) or drugs (unconditioned stimulus). Whether conditioned stimulus and unconditioned stimulus retrieval trigger different memory reconsolidation processes is not clear. Methods: Protein synthesis inhibitor or ß-adrenergic receptor (ß-AR) antagonist was systemically administrated or intra-central amygdala infused immediately after cocaine reexposure in cocaine-conditioned place preference or self-administration mice models. ß-ARs were selectively knocked out in the central amygdala to further confirm the role of ß-adrenergic receptor in cocaine reexposure-induced memory reconsolidation of cocaine-conditioned place preference. Results: Cocaine reexposure triggered de novo protein synthesis dependent memory reconsolidation of cocaine-conditioned place preference. Cocaine-priming-induced reinstatement was also impaired with post cocaine retrieval manipulation, in contrast to the relapse behavior with post context retrieval manipulation. Cocaine retrieval, but not context retrieval, induced central amygdala activation. Protein synthesis inhibitor or ß1-adrenergic receptor antagonist infused in the central amygdala after cocaine retrieval, but not context retrieval, inhibited memory reconsolidation and reinstatement. ß1-adrenergic receptor knockout in the central amygdala suppressed cocaine retrieval-triggered memory reconsolidation and reinstatement of cocaine conditioned place preference. ß1-adrenergic receptor antagonism after cocaine retrieval also impaired reconsolidation and reinstatement of cocaine self-administration. Conclusions: Cocaine reward memory triggered by unconditioned stimulus retrieval is distinct from conditioned stimulus retrieval. Unconditioned stimulus retrieval induced reconsolidation of cocaine reward memory depends on ß1-adrenergic signaling in the central amygdala. Post unconditioned stimulus retrieval manipulation can prevent drug memory reconsolidation and relapse to cocaine, thus providing a potential strategy for the prevention of substance addiction. Significance Statement: It is well known that drug memories become labile and reconsolidated upon retrieval by the presentation of conditioned stimulus (CS) or unconditioned stimulus (US). Whether CS and US retrieval trigger different memory reconsolidation processes is unknown. In this study, we found that US retrieval, but not CS retrieval, triggered memory reconsolidation of cocaine-conditioned place preference dependent on ß1-AR and de novo protein synthesis in the central amygdala. Furthermore, cocaine priming-induced reinstatement was impaired with post US retrieval manipulation in contrast to the relapse behavior with post CS retrieval manipulation. In cocaine self-administration, ß1-AR antagonism after US retrieval also impaired reconsolidation and reinstatement. Our study indicates that reconsolidation of cocaine reward memory triggered by US retrieval is distinct from CS retrieval. US retrieval induced reconsolidation of cocaine reward memory depends on ß1-adrenergic signaling in the central amygdala.

Unconditioned- and Conditioned- Stimuli Induce Differential Memory Reconsolidation and ß-AR-Dependent CREB Activation.

Consolidated long-term fear memories become labile and reconsolidated upon retrieval by the presentation of conditioned stimulus (CS) or unconditioned stimulus (US). Whether CS-retrieval or US-retrieval will trigger different memory reconsolidation processes is unknown. In this study, we introduced a sequential fear conditioning paradigm in which footshock (FS) was paired with two distinct sounds (CS-A and CS-B). The treatment with propranolol, a ß-adrenergic receptor (ß-AR) antagonist, after US (FS)-retrieval impaired freezing behavior evoked by either CS-A or CS-B. Betaxolol, a selective ß1-AR antagonist, showed similar effects. However, propranolol treatment after retrieval by one CS (e.g., CS-A) only inhibited freezing behavior evoked by the same CS (i.e., CS-A), not the other CS (CS-B). These data suggest that ß-AR is critically involved in reconsolidation of fear memory triggered by US- and CS-retrieval, whereas ß-AR blockade after US-retrieval disrupts more CS-US associations than CS-retrieval does. Furthermore, significant CREB activation in almost the whole amygdala and hippocampus was observed after US-retrieval, but CS-retrieval only stimulated CREB activation in the lateral amygdala and the CA3 of hippocampus. In addition, propranolol treatment suppressed memory retrieval-induced CREB activation. These data indicate that US-retrieval activates more memory traces than CS-retrieval does, leading to memory reconsolidation of more CS-US associations.

Drug-seeking motivation level in male rats determines offspring susceptibility or resistance to cocaine-seeking behaviour.

Liability to develop drug addiction is heritable, but the precise contribution of non-Mendelian factors is not well understood. Here we separate male rats into addiction-like and non-addiction-like groups, based on their incentive motivation to seek cocaine. We find that the high incentive responding of the F0 generation could be transmitted to F1 and F2 generations. Moreover, the inheritance of high incentive response to cocaine is contingent on high motivation, as it is elicited by voluntary cocaine administration, but not high intake of cocaine itself. We also find DNA methylation differences between sperm of addiction-like and non-addiction-like groups that were maintained from F0 to F1, providing an epigenetic link to transcriptomic changes of addiction-related signalling pathways in the nucleus accumbens of offspring. Our data suggest that highly motivated drug seeking experience may increase vulnerability and/or reduce resistance to drug addiction in descendants.

Response of soil microbial activities and microbial community structure to vanadium stress.

High levels of vanadium (V) have long-term, hazardous impacts on soil ecosystems and biological processes. In the present study, the effects of V on soil enzymatic activities, basal respiration (BR), microbial biomass carbon (MBC), and the microbial community structure were investigated through 12-week greenhouse incubation experiments. The results showed that V content affected soil dehydrogenase activity (DHA), BR, and MBC, while urease activity (UA) was less sensitive to V stress. The average median effective concentration (EC50) thresholds of V were predicted using a log-logistic dose-response model, and they were 362mgV/kg soil for BR and 417mgV/kg soil for DHA. BR and DHA were more sensitive to V addition and could be used as biological indicators for soil V pollution. According to a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, the structural diversity of the microbial community decreased for soil V contents ranged between 254 and 1104mg/kg after 1 week of incubation. As the incubation time increased, the diversity of the soil microbial community structure increased for V contents ranged between 354 and 1104mg/kg, indicating that some new V-tolerant bacterial species might have replicated under these conditions.

Lack of ß2-AR Increases Anxiety-Like Behaviors and Rewarding Properties of Cocaine.

It is well known that ß-adrenoceptors (ß-ARs) play a critical role in emotional arousal and stressful events, but the specific contributions of the ß2-AR subtype to the psychological disorders are largely unknown. To investigate whether ß2-AR are involved in anxiety-like behavior and reward to addictive drugs, we conducted a series of behavioral tests on ß2-AR knock-out (KO) mice. ß2-AR KO mice exhibited increased preference for the dark compartment and closed arm in tests of Light/Dark box and elevated plus maze, indicating that ß2-AR deletion elevates level of anxiety or innate fear. ß2-AR KO mice also showed decreased immobility in tail suspension test (TST), suggesting that ß2-AR deletion inhibits depression-like behavior. Interestingly, ß2-AR ablation did not change basal locomotion but significantly increased locomotor activity induced by acute cocaine administration. ß2-AR KO mice showed enhanced place preference for cocaine, which could be attenuated by ß1-selective AR antagonist betaxolol. Consistently, ß2-AR agonist suppressed cocaine-conditioned place preference (CPP). These data indicate that ß2-AR deletion enhances acute response and reward to cocaine. Our results suggest that ß2-AR regulates anxiety level, depression-like behavior and hedonic properties of cocaine, implicating that ß2-AR are the potential targets for the treatment of emotional disorders and cocaine addiction.

[Study on synergic and decreasing toxic effects of mineral water and Chinese herbal compound preparation on cisplatin].

OBJECTIVE: The synergic and decreasing toxic effects of mineral water and Chinese herbal compound preparation (MWCHCP) on cisplatin were investigated in sarcoma 180 (S180) mice. METHOD: The S180 mice were treated for 5 days with intraperitoneal injection of cisplatin(7.33 mg x kg(-1)) and oral administration of MWCHCP(1 925, 3 850, 7 700 mg x kg(-1)). Then the mice were killed and the tumor growth inhibition rate, organ index, diarrhea index were determined. Observe pathological sections of stomach to study the protective effect of MWCHCP. Reverse transcription-polymerase chain reaction (RT-PCR) was applied to investigate the tumour necrosis factor-alpha (TNF-alpha) expression level of the intestine. RESULT: Combining with cisplatin and MWCHCP caused a tendency of increasing the tumor growth inhibition rate and significant attenution of cisplatin-induced diarrhea, visceral organ injury, gastric mucosal injury and decreased TNF-alpha mRNA level of intestine. CONCLUSION: The present findings suggest that MWCHCP increases the inhibition rate of tumor growth of cisplatin and has a beneficial influence on gastrointestinal lesion induced by cisplatin.