Contribution to net zero emissions of integrating hydrogen production in wastewater treatment plants.

The reliability of renewable hydrogen supply for off-take applications is critical to the future sustainable energy economy. Integrated water electrolysis can be deployed at distributed municipal wastewater treatment plants (WWTP), creating opportunity for reduction in carbon emissions through direct and indirect use of the electrolysis output. A novel energy shifting process where the co-produced oxygen is compressed and stored to enhance the utilisation of intermittent renewable electricity is analysed. The hydrogen produced can be used in local fuel cell electric buses to replace incumbent diesel buses for public transport. However, quantifying the extent of carbon emission reduction of this conceptual integrated system is key. In this study, the integration of hydrogen production at a case study WWTP of 26,000 EP capacity and using the hydrogen in buses was compared with two conventional systems: the base case of a WWTP with grid electricity consumption offset by solar PV and the community's independent use of diesel buses for transport, and the non-integrated configuration with hydrogen produced at the bus refuelling location operated independently of the WWTP. The system response was analysed using a Microsoft Excel simulation model with hourly time steps over a 12-month time frame. The model included a control scheme for the reliable supply of hydrogen for public transport and oxygen to the WWTP, and considered expected reductions in carbon intensity of the national grid, level of solar PV curtailment, electrolyser efficiency and size of the solar PV system. Results showed that by 2031, when Australia's national electricity is forecast to achieve a carbon intensity of less than 0.186 kg CO2-e/kWh, integrating water electrolysis at a municipal WWTP for producing hydrogen for use in local hydrogen buses produced less carbon emissions than continuing to use diesel buses and offsetting emissions by exporting renewable electricity to the grid. By 2034, an annual reduction of 390 t-CO2-e is expected after changing to the integrated configuration. Considering electrolyser efficiency improvements and curtailment of renewable electricity, the reduction increases to 872.8 t-CO2-e.

Energy shifting in wastewater treatment using compressed oxygen from integrated hydrogen production.

Integrating renewable hydrogen production via electrolysis with wastewater treatment is an opportunity to manage environmental resources more sustainably while providing a pathway to producing sustainable hydrogen at industrial scale. The synergies of integrating oxygen production from water electrolysis and oxygen use in wastewater treatment benefit both hydrogen production and wastewater industries. However, the understanding of the most suitable integrated process configuration and scale of renewable equipment is not known. A novel energy shifting process is proposed here using compressed and stored oxygen produced by water electrolysis and used in the activated sludge process, replacing traditional aeration in the wastewater treatment plant and eliminating the high energy consuming blowers supplying air to submerged fine bubble diffusers. In the proposed energy shifting process, excess oxygen produced by water electrolysis at times of peak renewable electricity production is stored and used for wastewater treatment at times of peak oxygen demand. Wastewater treatment data from the activated sludge process was used to calculate oxygen demand in 1-h intervals over a 24-h period, and the system response of the integrated plant was simulated at hourly intervals and equipment size determined according to an optimisation algorithm that balances oxygen and electricity supply and demand over a 24-h period. Sensitivity analysis of operational parameters is assessed and the case for replacing traditional WWTP aeration with newer technologies is quantified using a high efficiency oxygen transfer system such as a Speece cone as an example that is shown to be a prerequisite for the feasibility of the process. The results produced by this study provide valuable information to the hydrogen and wastewater industries on how an integrated plant could be configured. Besides the environmental advantages of sustainably produced hydrogen, using oxygen as a biochemical energy storage medium in this configuration means WWTP powered from renewable electricity becomes more viable reducing the industries reliance on fossil fuels.

Recognition and management of clozapine adverse effects: A systematic review and qualitative synthesis.

OBJECTIVE: Clozapine is substantially underutilized in most countries and clinician factors including lack of knowledge and concerns about adverse drug effects (ADEs) contribute strongly to treatment reluctance. The aim of this systematic review was to provide clinicians with a comprehensive information source regarding clozapine ADEs. METHODS: PubMed and Embase databases were searched for English language reviews concerned with clozapine ADEs; publications identified by the automated search were manually searched for additional relevant citations. Following exclusion of redundant and irrelevant reports, pertinent information was summarized in evidence tables corresponding to each of six major ADE domains; two authors reviewed all citations for each ADE domain and summarized their content by consensus in the corresponding evidence table. This study was conducted in accordance with PRISMA principles. RESULTS: Primary and secondary searches identified a total of 305 unique reports, of which 152 were included in the qualitative synthesis. Most clozapine ADEs emerge within 3 months, and almost all appear within 6 months, after initiation. Notable exceptions are weight gain, diabetic ketoacidosis (DKA), severe clozapine-induced gastrointestinal hypomotility (CIGH), clozapine-induced cardiomyopathy (CICM), seizures, and clozapine-induced neutropenia (CIN). Most clozapine ADEs subside gradually or respond to dose reduction; those that prompt discontinuation generally do not preclude rechallenge. Rechallenge is generally inadvisable for clozapine-induced myocarditis (CIM), CICM, and clozapine-induced agranulocytosis (CIA). Clozapine plasma levels >600-1000 µg/L appear more likely to cause certain ADEs (e.g., seizures) and, although there is no clear toxicity threshold, risk/benefit ratios are generally unfavorable above 1000 µg/L. CONCLUSION: Clozapine ADEs rarely require discontinuation.

Organic nitrogen nutrition: LHT1.2 protein from hybrid aspen (Populus tremula L. x tremuloides Michx) is a functional amino acid transporter and a homolog of Arabidopsis LHT1.

The contribution of amino acids (AAs) to soil nitrogen (N) fluxes is higher than previously thought. The fact that AA uptake is pivotal for N nutrition in boreal ecosystems highlights plant AA transporters as key components of the N cycle. At the same time, very little is known about AA transport and respective transporters in trees. Tree genomes may contain 13 or more genes encoding the lysine histidine transporter (LHT) family proteins, and this complicates the study of their significance for tree N-use efficiency. With the strategy of obtaining a tool to study N-use efficiency, our aim was to identify and characterize a relevant AA transporter in hybrid aspen (Populus tremula L. x tremuloides Michx.). We identified PtrLHT1.2, the closest homolog of Arabidopsis thaliana (L.) Heynh AtLHT1, which is expressed in leaves, stems and roots. Complementation of a yeast AA uptake mutant verified the function of PtrLHT1.2 as an AA transporter. Furthermore, PtrLHT1.2 was able to fully complement the phenotypes of the Arabidopsis AA uptake mutant lht1 aap5, including early leaf senescence-like phenotype, reduced growth, decreased plant N levels and reduced root AA uptake. Amino acid uptake studies finally showed that PtrLHT1.2 is a high affinity transporter for neutral and acidic AAs. Thus, we identified a functional AtLHT1 homolog in hybrid aspen, which harbors the potential to enhance overall plant N levels and hence increase biomass production. This finding provides a valuable tool for N nutrition studies in trees and opens new avenues to optimizing tree N-use efficiency.

Activating Iron Based Materials for Overall Electrochemical Water Splitting via the Incorporation of Noble Metals.

Although the presence of iron in mixed metal oxide based catalysts has shown significant performance improvement in the oxygen evolution reaction (OER), iron oxides themselves demonstrate much poorer activity. In this study, we investigate improving the performance of iron catalysts via surface decoration with gold or platinum for not only the OER but also the hydrogen evolution reaction (HER) for overall water splitting in an alkaline electrolyte. Two types of iron catalysts were synthesised, iron nanocubes and iron oxide via electrochemical deposition methods which were decorated with either Au or Pt via galvanic replacement. It was found that the presence of Au significantly enhanced the OER performance of iron oxide and the HER performance of iron nanocubes. The presence of Pt resulted in moderate improvement in the OER but significant improvement for the HER but did not surpass the performance of gold decorated iron nanocubes. This indicates that the speciation of the iron catalyst and the decorating metal was important for tuning the activity to the OER and the HER. For the OER, the formation of iron oxide/Au interfaces was determined to be an important component for high activity whereas the metallic nature of metal decorated iron nanocubes was important for the HER. Therefore, iron based catalysts can be modified to demonstrate bifunctional behaviour for overall water splitting via the inclusion of gold nanoparticles.

Ethylene Signaling Is Required for Fully Functional Tension Wood in Hybrid Aspen.

Tension wood (TW) in hybrid aspen trees forms on the upper side of displaced stems to generate a strain that leads to uplifting of the stem. TW is characterized by increased cambial growth, reduced vessel frequency and diameter, and the presence of gelatinous, cellulose-rich (G-)fibers with its microfibrils oriented parallel to the fiber cell axis. Knowledge remains limited about the molecular regulators required for the development of this special xylem tissue with its characteristic morphological, anatomical, and chemical features. In this study, we use transgenic, ethylene-insensitive (ETI) hybrid aspen trees together with time-lapse imaging to show that functional ethylene signaling is required for full uplifting of inclined stems. X-ray diffraction and Raman microspectroscopy of TW in ETI trees indicate that, although G-fibers form, the cellulose microfibril angle in the G-fiber S-layer is decreased, and the chemical composition of S- and G-layers is altered than in wild-type TW. The characteristic asymmetric growth and reduction of vessel density is suppressed during TW formation in ETI trees. A genome-wide transcriptome profiling reveals ethylene-dependent genes in TW, related to cell division, cell wall composition, vessel differentiation, microtubule orientation, and hormone crosstalk. Our results demonstrate that ethylene regulates transcriptional responses related to the amount of G-fiber formation and their properties (chemistry and cellulose microfibril angle) during TW formation. The quantitative and qualitative changes in G-fibers are likely to contribute to uplifting of stems that are displaced from their original position.

Genotypic variation in Pinus radiata responses to nitrogen source are related to changes in the root microbiome.

Variation in traits within a plant species contributes to differences in soil physicochemistry and rhizosphere microbial communities. However, how intraspecific variation in plant responses to nitrogen (N) shapes these communities remains unclear. We studied whether plant responses to organic and inorganic N forms vary among genotypes, and if these responses were associated with variation in root-associated communities. We investigated how the root microbiomes of two Pinus radiata D. Don genotypes were altered by two years of N-fertilisation in field conditions. We characterised rhizosphere bacterial and fungal communities, as well as root-associated fungal communities, of trees receiving yearly additions of NH4NO3 or L-arginine, and control trees. We also measured plant traits and rhizosphere soil physicochemical properties. Two main findings emerged: (i) N form and tree genotype affected soil physicochemical properties as well as plant measures, and these responses were associated with variation in microbial communities, and (ii) rhizosphere and root-associated communities differed in their responses to N form and host genotype. Our results suggest that N forms have different influences on N and carbon dynamics at the plant-soil interface by inducing root-mediated responses that are associated with shifts in the root microbiome such that communities more closely associated with roots are more sensitive to genotype-specific responses.

Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen.

The phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. We report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation. We applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild-type and ethylene-insensitive hybrid aspen trees (Populus tremula × tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild-type and ethylene-insensitive trees. We demonstrate that ACC and ethylene induce gelatinous layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (Ethylene Response Factors (ERFs), ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1)) and wood formation. G-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

Host Genotype and Nitrogen Form Shape the Root Microbiome of Pinus radiata.

A central challenge in community ecology is understanding the role that phenotypic variation among genotypes plays in structuring host-associated communities. While recent studies have investigated the relationship between plant genotype and the composition of soil microbial communities, the effect of genotype-by-environment interactions on the plant microbiome remains unclear. In this study, we assessed the influence of tree genetics (G), nitrogen (N) form and genotype-by-environment interaction (G x N) on the composition of the root microbiome. Rhizosphere communities (bacteria and fungi) and root-associated fungi (including ectomycorrhizal and saprotrophic guilds) were characterised in two genotypes of Pinus radiata with contrasting physiological responses to exogenous organic or inorganic N supply. Genotype-specific responses to N form influenced the composition of the root microbiome. Specifically, (1) diversity and composition of rhizosphere bacterial and root-associated fungal communities differed between genotypes that had distinct responses to N form, (2) shifts in the relative abundance of individual taxa were driven by the main effects of N form or host genotype and (3) the root microbiome of the P. radiata genotype with the most divergent growth responses to organic and inorganic N was most sensitive to differences in N form. Our results show that intraspecific variation in tree response to N form has significant consequences for the root microbiome of P. radiata, demonstrating the importance of genotype-by-environment interactions in shaping host-associated communities.

Depletion of carbohydrate reserves limits nitrate uptake during early regrowth in Lolium perenne L.

The mechanisms linking C/N balance to N uptake and assimilation are central to plant responses to changing soil nutrient levels. Defoliation and subsequent regrowth of grasses both impact C partitioning, thereby creating a significant point of interaction with soil N availability. Using defoliation as an experimental treatment, we investigated the dynamic relationships between plant carbohydrate status and NO3--responsive uptake systems, transporter gene expression, and nitrate assimilation in Lolium perenne L. High- and low-affinity NO3- uptake was reduced in an N-dependent manner in response to a rapid and large shift in carbohydrate remobilization triggered by defoliation. This reduction in NO3- uptake was rescued by an exogenous glucose supplement, confirming the carbohydrate dependence of NO3- uptake. The regulation of NO3- uptake in response to the perturbation of the plant C/N ratio was associated with changes in expression of putative high- and low-affinity NO3- transporters. Furthermore, NO3- assimilation appears to be regulated by the C-N status of the plant, implying a mechanism that signals the availability of C metabolites for NO3- uptake and assimilation at the whole-plant level. We also show that cytokinins may be involved in the regulation of N acquisition and assimilation in response to the changing plant C/N ratio.

Coordinated nitrogen and carbon remobilization for nitrate assimilation in leaf, sheath and root and associated cytokinin signals during early regrowth of Lolium perenne.

Background and Aims: The efficiency of N assimilation in response to defoliation is a critical component of plant regrowth and forage production. The aim of this research was to test the effect of the internal C/N balance on NO3- assimilation and to estimate the associated cytokinin signals following defoliation of perennial ryegrass ( Lolium perenne L. 'Grasslands Nui') plants. Methods: Plants, manipulated to have contrasting internal N content and contrasting availability of water soluble carbohydrates (WSCs), were obtained by exposure to either continuous light or short days (8:16 h light-dark), and watered with modified N-free Hoagland medium containing either high (5 m m ) or low (50 µ m ) NO3- as sole N source. Half of the plants were defoliated and the root, sheath and leaf tissue were harvested at 8, 24 and 168 h after cutting. The spatiotemporal changes in WSCs, synthesis of amino acids and associated cytokinin content were recorded after cutting. Key Results: Leaf regrowth following defoliation involved changes in the low- and high-molecular weight WSCs. The extent of the changes and the partitioning of the WSC following defoliation were dependant on the initial WSC levels and the C and N availability. Cytokinin levels varied in the sheath and root as early as 8 h following defoliation and preceded an overall increase in amino acids at 24 h. Subsequently, negative feedback brought the amino acid response back towards pre-defoliation levels within 168 h after cutting, a response that was under control of the C/N ratio. Conclusions: WSC remobilization in the leaf is coordinated with N availability to the root, potentially via a systemic cytokinin signal, leading to efficient N assimilation in the leaf and the sheath tissues and to early leaf regrowth following defoliation.

Insights into the functional relationship between cytokinin-induced root system phenotypes and nitrate uptake in Brassica napus.

Root system architecture is the spatial arrangement of roots that impacts the capacity of plants to access nutrients and water. We employed pharmacologically generated morphological and molecular phenotypes and used in situ 15N isotope labelling, to investigate whether contrasting root traits are of functional interest in relation to nitrate acquisition. Brassica napus L. were grown in solidified phytogel culture media containing 1mM KNO3 and treated with the cytokinin, 6-benzylaminopurine, the cytokinin antagonist, PI-55, or both in combination. The pharmacological treatments inhibited root elongation relative to the control. The contrasting root traits induced by PI-55 and 6-benzylaminopurine were strongly related to 15N uptake rate. Large root proliferation led to greater 15N cumulative uptake rather than greater 15N uptake efficiency per unit root length, due to a systemic response in the plant. This relationship was associated with changes in C and N resource distribution between the shoot and root, and in expression of BnNRT2.1, a nitrate transporter. The root:shoot biomass ratio was positively correlated with 15N cumulative uptake, suggesting the functional utility of root investment for nutrient acquisition. These results demonstrate that root proliferation in response to external nitrate is a behaviour which integrates local N availability and the systemic N status of the plant.

Metabolic changes and associated cytokinin signals in response to nitrate assimilation in roots and shoots of Lolium perenne.

The efficiency of inorganic nitrogen (N) assimilation is a critical component of fertilizer use by plants and of forage production in Lolium perenne, an important pasture species worldwide. We present a spatiotemporal description of nitrate use efficiency in terms of metabolic responses and carbohydrate remobilization, together with components of cytokinin signal transduction following nitrate addition to N-impoverished plants. Perennial ryegrass (L. perenne cv. Grasslands Nui) plants were grown for 10 weeks in unfertilized soil and then treated with nitrate (5 mM) hydroponically. Metabolomic analysis by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry revealed a dynamic interaction between N and carbon metabolism over a week-long time course represented by the relative abundance of amino acids, tricarboxylic acid intermediates and stored water-soluble carbohydrates (WSCs). The initial response to N addition was characterized by a rapid remobilization of carbon stores from the low-molecular weight WSC, along with an increase in N content and assimilation into free amino acids. Subsequently, the shoot became the main source of carbon through remobilization of a large pool of high-molecular weight WSC. Associated quantification of cytokinin levels and expression profiling of putative cytokinin response regulator genes by quantitative reverse transcription polymerase chain reaction support a role for cytokinin in the mediation of the response to N addition in perennial ryegrass. The presence of high levels of cis-zeatin-type cytokinins is discussed in the context of hormonal homeostasis under the stress of steady-state N deficiency.

Decreasing mortality from acute biliary diseases that require endoscopic retrograde cholangiopancreatography: a nationwide cohort study.

BACKGROUND & AIMS: The management of acute biliary diseases often involves endoscopic retrograde cholangiopancreatography (ERCP), but it is not clear whether this technique reduces mortality. We investigated whether mortality from acute biliary diseases that require ERCP has been reduced over time and explored factors associated with mortality. METHODS: We conducted a cohort study using the Nationwide Inpatient Sample (1998-2008). We identified hospitalizations for choledocholithiasis, cholangitis, and acute pancreatitis that involved ERCP. Multivariate analyses were used to determine the effects of time period, patient factors, hospital characteristics, features of the ERCP procedure, and types of cholecystectomies on mortality, length of stay, and costs. RESULTS: From 1998 to 2008 there were 166,438 admissions for acute biliary conditions that met the inclusion criteria, corresponding to more than 800,000 patients nationwide. During this interval, mortality decreased from 1.1% to 0.6% (adjusted odds ratio [aOR], 0.7; 95% confidence interval [CI], 0.6-0.8), diagnostic ERCPs decreased from 28.8% to 10.0%, hospitals performing fewer than 100 ERCPs per year decreased from 38.4% to 26.9%, open cholecystectomies decreased from 12.4% to 5.8%, and unsuccessful ERCPs decreased from 6.3% to 3.2% (P < .0001 for all trends). Unsuccessful ERCP (aOR, 1.7; 95% CI, 1.4-2.2), open cholecystectomy (aOR, 3.4; 95% CI 2.7-4.3), cholangitis (aOR, 1.9; 95% CI, 1.5-2.3), older age, having Medicare health insurance, and comorbidity were associated with increased mortality. CONCLUSIONS: In-hospital mortality from acute biliary conditions requiring ERCP in the United States has decreased over time. Reductions in the rate of unsuccessful ERCPs and open cholecystectomies are associated with this trend.

Bile acid malabsorption in chronic diarrhea: pathophysiology and treatment.

BACKGROUND: Bile acid malabsorption (BAM) is a common but frequently under-recognized cause of chronic diarrhea, with an estimated prevalence of 4% to 5%. METHODS: The published literature for the period 1965 to 2012 was examined for articles regarding the pathophysiology and treatment of BAM to provide an overview of the management of BAM in gastroenterology practice. RESULTS: BAM is classified as type 1 (secondary to ileal dysfunction), type 2 (idiopathic) or type 3 (secondary to gastrointestinal disorders not associated with ileal dysfunction). The estimated prevalence of BAM is >90% in patients with resected Crohn disease (CD) and 11% to 52% of unresected CD patients (type 1); 33% in diarrhea-predominant irritable bowel syndrome (type 2); and is a frequent finding postcholecystectomy or postvagotomy (type 3). Investigations include BAM fecal bile acid assay, 23-seleno-25-homo-tauro-cholic acid (SeHCAT) testing and high-performance liquid chromatography of serum 7-α-OH-4-cholesten-3-one (C4), to determine the level of bile acid synthesis. A less time-consuming and expensive alternative in practice is an empirical trial of the bile acid sequestering agent cholestyramine. An estimated 70% to 96% of chronic diarrhea patients with BAM respond to short-course cholestyramine. Adverse effects include constipation, nausea, borborygmi, flatulence, bloating and abdominal pain. Other bile acid sequestering agents, such as colestipol and colesevelam, are currently being investigated for the treatment of BAM-associated diarrhea. CONCLUSIONS: BAM is a common cause of chronic diarrhea presenting in gastroenterology practice. In accordance with current guidelines, an empirical trial of a bile acid sequestering agent is warranted as part of the clinical workup to rule out BAM.

A genome-wide screen for ethylene-induced ethylene response factors (ERFs) in hybrid aspen stem identifies ERF genes that modify stem growth and wood properties.

Ethylene Response Factors (ERFs) are a large family of transcription factors that mediate responses to ethylene. Ethylene affects many aspects of wood development and is involved in tension wood formation. Thus ERFs could be key players connecting ethylene action to wood development. We identified 170 gene models encoding ERFs in the Populus trichocarpa genome. The transcriptional responses of ERF genes to ethylene treatments were determined in stem tissues of hybrid aspen (Populus tremula × tremuloides) by qPCR. Selected ethylene-responsive ERFs were overexpressed in wood-forming tissues and characterized for growth and wood chemotypes by FT-IR. Fifty ERFs in Populus showed more than five-fold increased transcript accumulation in response to ethylene treatments. Twenty-six ERFs were selected for further analyses. A majority of these were induced during tension wood formation. Overexpression of ERFs 18, 21, 30, 85 and 139 in wood-forming tissues of hybrid aspen modified the wood chemotype. Moreover, overexpression of ERF139 caused a dwarf-phenotype with altered wood development, and overexpression of ERF18, 34 and 35 slightly increased stem diameter. We identified ethylene-induced ERFs that respond to tension wood formation, and modify wood formation when overexpressed. This provides support for their role in ethylene-mediated regulation of wood development.

Summary of guidelines for infection prevention and control for flexible gastrointestinal endoscopy.

BACKGROUND: High-quality processes to ensure infection prevention and control in the delivery of safe endoscopy services are essential. In 2010, the Public Health Agency of Canada and the Canadian Association of Gastroenterology (CAG) developed a Canadian guideline for the reprocessing of flexible gastrointestinal endoscopy equipment. METHODS: The CAG Endoscopy Committee carefully reviewed the 2010 guidelines and prepared an executive summary. RESULTS: Key elements relevant to infection prevention and control for flexible gastrointestinal endoscopy were highlighted for each of the recommendations included in the 2010 document. The 2010 guidelines consist of seven sections, including administrative recommendations, as well as recommendations for endoscopy and endoscopy decontamination equipment, reprocessing endoscopes and accessories, endoscopy unit design, quality management, outbreak investigation and management, and classic and variant Creutzfeldt-Jakob Disease. DISCUSSION: The recommendations for infection prevention and control for flexible gastrointestinal endoscopy are intended for all individuals with responsibility for endoscopes in all settings where endoscopy is performed.