Cradle to gate: life cycle impact of primary aluminium production

Purpose

The International Aluminium Institute’s (IAI) aim was to publish life cycle inventory (LCI) data for use by life cycle assessment (LCA) practitioners through professional databases. The need to provide robust data stems from the increasing application of LCA as a tool for making material and design choices and the importance for representative, up-to-date information to underpin such studies. In addition to this, the institute aimed to evaluate the significance of potential environmental impacts, based on the LCI results, against a defined set of impact categories which can be tracked over time.

Methods

Key environmental data collected as part of the IAI’s long-running industry surveys provided the foundation for the life cycle inventory. In order to evaluate the environmental impact, direct input and output data for primary aluminium production were supplemented with background data for indirect processes available in GaBi version 6 (PE International, 2013b). A cradle-to-gate model was constructed with two distinct datasets, global (GLO) and global minus China (rest of world (RoW)). A partial life cycle impact assessment (LCIA) was completed using the models, and the following six CML (2001–Nov 2010) midpoint environmental impact categories were reported: acidification potential, depletion of fossil energy resources, eutrophication potential, global warming potential, ozone depletion potential and photo-oxidant creation potential. Water scarcity footprint of primary aluminium (Buxmann et al. in this issue) was also included.

Results and discussion

The results indicated that the largest greenhouse gas contributions were attributed to the alumina refining and electrolysis unit processes in both datasets, with electricity and thermal energy, being the major contributing factors to these higher values. The energy intensive nature of primary aluminium production means energy supply can significantly influence the overall environmental impact. Electricity production was found to contribute between 25 % and 80 % to all impact category indicator results, with higher values in the global dataset, a result of the inclusion of Chinese energy data and the increased share of coal-based electricity consumption that it represents.

Conclusions

The global aluminium industry remains dedicated to transparent reporting of its environmental impacts and ensuring that up-to-date, representative LCI data is available. Development of suitable methodologies for new indicators will be required to ensure that the industry continues to report accurately all its relevant impacts. Additionally, with the increased importance of Chinese aluminium production, inclusion of foreground data from Chinese production would further enhance the dataset from which the global impacts of aluminium production are assessed from cradle to gate.

     

Consistent characterisation factors at midpoint and endpoint relevant to agricultural water scarcity arising from freshwater consumption

Purpose

The shortage of agricultural water from freshwater sources is a growing concern because of the relatively large amounts needed to sustain food production for an increasing population. In this context, an impact assessment methodology is indispensable for the identification and assessment of the potential consequences of freshwater consumption in relation to agricultural water scarcity. This paper reports on the consistent development of midpoint and endpoint characterisation factors (CFs) for assessing these impacts.

Methods

Midpoint characterisation factors focus specifically on shortages in food production resulting from agricultural water scarcity. These were calculated by incorporating country-specific compensation factors for physical availability of water resources and socio-economic capacity in relation to the irrigation water demand for agriculture. At the endpoint, to reflect the more complex impact pathways from food production losses to malnutrition damage from agricultural water scarcity, international food trade relationships and economic adaptation capacity were integrated in the modelling with measures of nutritional vulnerability for each country.

Results and discussion

The inter-country variances of CFs at the midpoint revealed by this study were larger than those derived using previously developed methods, which did not integrate compensation processes by food stocks. At the endpoint level, both national and trade-induced damage through international trade were quantified and visualised. Distribution of malnutrition damage was also determined by production and trade balances for commodity groups in water-consuming countries, as well as dependency on import ratios for importer countries and economic adaptation capacity in each country. By incorporating the complex relationships between these factors, estimated malnutrition damage due to freshwater consumption at the country scale showed good correlation with total reported nutritional deficiency damage.

Conclusions

The model allows the establishment of consistent CFs at the midpoint and endpoint for agricultural water scarcity resulting from freshwater consumption. The complex relationships between food production supply and nutrition damage can be described by considering the physical and socio-economic parameters used in this study. Developed CFs contribute to a better assessment of the potential impacts associated with freshwater consumption in global supply chains and to life cycle assessment and water footprint assessments.

     

Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease

Background

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients.

Methods

Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE^?/?) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients.

Results

Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r?=?0.520, p <?0.0001).

Conclusion

Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted.

     

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

Background

Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis.

Methods

Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing.

Results

The main features and advantages of this protocol are:

• An optimized method for extracting good quality DNA from FFPE tissues.
• An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue.
• Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing.

Conclusions

We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

     

Analysis of greenhouse gas emissions related to aluminium transport applications

Background, aim and scope

Climate change is a subject of growing global concern. Based on International Energy Agency (IEA 2004) research, about 19% of the greenhouse gas emissions from fuel combustion are generated by the transportation sector, and its share is likely to grow. Significant increases in the vehicles fleets are expected in particular in China, India, the Middle East and Latin America. As a result, reducing vehicle fuel consumption is most essential for the future. The reduction of the vehicle weight, the introduction of improved engine technologies, lower air friction, better lubricants, etc. are established methods of improving fuel efficiency, reducing energy consumption and greenhouse gas emissions. Continued progress will be required along all these fronts with light-weighting being one of the most promising options for the global transport sector. This paper quantifies greenhouse gas savings realised from light-weighting cars with aluminium based on life cycle assessment methodology. The study uses a pragmatic approach to assess mass reduction by comparing specific examples of components meeting identical performance criteria. The four examples presented in this analysis come from practical applications of aluminium. For each case study, the vehicle manufacturer has supplied the respective masses of the aluminium and the alternative component.

Material and methods

A full life cycle assessment with regards to greenhouse gas emissions and savings has been carried out for different aluminium applications in cars as compared to the same applications in steel or cast iron. The case studies reference real cases, where aluminium is actually used in series production. The studies are based on a greenhouse gas lifecycle model, which has been developed following the ISO standard 14040 framework. For each component, sensitivity analysis is applied to determine the impact of lifetime driving distance, driving characteristics (impact of air friction) and recycling rate.

Results

Life cycle results show that in automotive applications, each kilogram of aluminium replacing mild steel, cast iron or high strength steel saves, depending on the specific case (bumper and motor block of a compact car, front hood of a large family car, body-in white of a luxury car), between 13 and 20 kg of greenhouse gas emissions.

Discussion

The performed sensitivity analysis finds that even with ‘worst case’ scenarios savings are still significant.

Conclusions

The results not only demonstrate significant benefits of aluminium with regard to greenhouse gas savings but also show that these are very sensitive to variations of the recycling rate, the life-time driving distance and the driving behaviour.

Recommendations and perspectives

Good care is needed to gather life-cycle data and to make informed estimates, where no data are available. Furthermore, greenhouse gas savings for additional components should be calculated using this life cycle model to sustain the findings.

     

Global guidance on environmental life cycle impact assessment indicators: impacts of climate change,fine particulate matter formation,water consumption and land use

Purpose

Guidance is needed on best-suited indicators to quantify and monitor the man-made impacts on human health, biodiversity and resources. Therefore, the UNEP-SETAC Life Cycle Initiative initiated a global consensus process to agree on an updated overall life cycle impact assessment (LCIA) framework and to recommend a non-comprehensive list of environmental indicators and LCIA characterization factors for (1) climate change, (2) fine particulate matter impacts on human health, (3) water consumption impacts (both scarcity and human health) and 4) land use impacts on biodiversity.

Methods

The consensus building process involved more than 100 world-leading scientists in task forces via multiple workshops. Results were consolidated during a 1-week Pellston Workshop? in January 2016 leading to the following recommendations.

Results and discussion

LCIA framework: The updated LCIA framework now distinguishes between intrinsic, instrumental and cultural values, with disability-adjusted life years (DALY) to characterize damages on human health and with measures of vulnerability included to assess biodiversity loss. Climate change impacts: Two complementary climate change impact categories are recommended: (a) The global warming potential 100 years (GWP 100) represents shorter term impacts associated with rate of change and adaptation capacity, and (b) the global temperature change potential 100 years (GTP 100) characterizes the century-scale long term impacts, both including climate-carbon cycle feedbacks for all climate forcers. Fine particulate matter (PM_2.5) health impacts: Recommended characterization factors (CFs) for primary and secondary (interim) PM_2.5 are established, distinguishing between indoor, urban and rural archetypes. Water consumption impacts: CFs are recommended, preferably on monthly and watershed levels, for two categories: (a) The water scarcity indicator “AWARE” characterizes the potential to deprive human and ecosystems users and quantifies the relative Available WAter REmaining per area once the demand of humans and aquatic ecosystems has been met, and (b) the impact of water consumption on human health assesses the DALYs from malnutrition caused by lack of water for irrigated food production. Land use impacts: CFs representing global potential species loss from land use are proposed as interim recommendation suitable to assess biodiversity loss due to land use and land use change in LCA hotspot analyses.

Conclusions

The recommended environmental indicators may be used to support the UN Sustainable Development Goals in order to quantify and monitor progress towards sustainable production and consumption. These indicators will be periodically updated, establishing a process for their stewardship.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

A lost opportunity for science: journals promote data sharing in metabolomics but do not enforce it

Introduction

Data sharing is being increasingly required by journals and has been heralded as a solution to the ‘replication crisis’.

Objectives

(i) Review data sharing policies of journals publishing the most metabolomics papers associated with open data and (ii) compare these journals’ policies to those that publish the most metabolomics papers.

Methods

A PubMed search was used to identify metabolomics papers. Metabolomics data repositories were manually searched for linked publications.

Results

Journals that support data sharing are not necessarily those with the most papers associated to open metabolomics data.

Conclusion

Further efforts are required to improve data sharing in metabolomics.

     

Untargeted metabolomics suffers from incomplete raw data processing

Introduction

Untargeted metabolomics is a powerful tool for biological discoveries. To analyze the complex raw data, significant advances in computational approaches have been made, yet it is not clear how exhaustive and reliable the data analysis results are.

Objectives

Assessment of the quality of raw data processing in untargeted metabolomics.

Methods

Five published untargeted metabolomics studies, were reanalyzed.

Results

Omissions of at least 50 relevant compounds from the original results as well as examples of representative mistakes were reported for each study.

Conclusion

Incomplete raw data processing shows unexplored potential of current and legacy data.

     

Freshwater use analysis of cassava for food feed fuel in the Mun River basin,Thailand

Purpose

This research aims to assess the current freshwater use in the cassava supply chain for food, feed fuel in the Mun basin, and the water scarcity impact and possible options to increase cassava production to meet the future demand following the Renewable and Alternative Energy Development Plan (AEDP) target.

Methods

This research analyzes freshwater use based on ISO 14046 water footprint assessment. The analysis was implemented based on a life cycle perspective that determines the impact on freshwater use from cassava products along their supply chain. Both direct water use and indirect water use that associated are analyzed. Midpoint impact of water use was assessed using water stress index (WSI) to calculate water scarcity footprint.

Results and discussion

The results show that in the current situation, total freshwater use of all cassava-related product in Mun basin in the base case is 1140 million m³/year. When WSI was applied, water scarcity footprint of all cassava-related products in the Mun basin in the base case was only 147 million m³/year. In the scenario 1, increasing irrigation to increase yield in the existing cassava cultivation area in the Mun basin has the largest water use compare to other scenarios. Scenarios 2 and 3, expanding cassava cultivation area in Mun basin and in other regions, have lower water and water scarcity impact than scenario 1. The benefit from transforming paddy rice (in unsuitable areas) to cassava cultivation was also good. However, more resources are required including land, energy, or fertilizer, and other environmental impacts such as greenhouse gas emission or eutrophication could be increased from the increasing resource use. Therefore, the decision-making process needs to consider the trade-off between those factors, and a more complete life cycle assessment (LCA) on the envisioned alternatives should be applied for further analysis.

Conclusions

The increasing demand of biofuels derived from cassava can increase stress on water in the Mun River basin. Increasing irrigation water use in the area as per requirement could possibly increase yield to meet the future feedstock demand but has large water scarcity impact. However, this could be alleviated by using groundwater from additional wells in the farm. Expanding cassava cultivation area could be another option having low water scarcity impact, but it requires more resources and could increase other environmental impacts that need to be further analyzed by a complete LCA.

     

Junctional kyphosis: how can we detect and monitor it during growth?

Background

Despite its importance in affecting adult pain, and disability, there is a lack of universal criteria for the diagnosis and evaluation of thoraco-lumbar Junctional Kyphosis (JK) and a gold standard measurement and diagnostic system does not exist.This study aims to verify the sensibility and specificity of clinical, and Formetric surface topography (FST) data in identifying Junctional Kyphosis in respect to the radiographical standard references.

Methods

Design: This is a cross sectional study from a prospective database started in March 2003.Participants: 38 subjects.

Inclusion criteria: Patients selected by age according to Risser score 1, at first visit with lateral x-rays and FST. Diagnostic test used to detect JK:

• FST criteria: level of thoraco-lumbar inflexion point in percentage compared to the total height of the spine.
• X-ray criteria: lower limit of thoracic kyphosis below T12.

Statistics: sensitivity, specificity, positive (PPV) and negative predictive values (NPV), ROC curve.

Results

FST showed a good reliability in detecting JK: with a threshold of 75 %, PPV was 100 %, NPV was 86 % and the Area Under the Curve was 83 %.

Conclusion

The need for a useful criteria able to characterize JK to allow diagnosis and monitoring of the deformity is still lacking, and further studies will deepen this issue.

     

A methodology for elucidating regulatory mechanisms leading to changes in lipid profiles

Introduction

It is difficult to elucidate the metabolic and regulatory factors causing lipidome perturbations.

Objectives

This work simplifies this process.

Methods

A method has been developed to query an online holistic lipid metabolic network (of 7923 metabolites) to extract the pathways that connect the input list of lipids.

Results

The output enables pathway visualisation and the querying of other databases to identify potential regulators. When used to a study a plasma lipidome dataset of polycystic ovary syndrome, 14 enzymes were identified, of which 3 are linked to ELAVL1—an mRNA stabiliser.

Conclusion

This method provides a simplified approach to identifying potential regulators causing lipid-profile perturbations.

     

KniMet: a pipeline for the processing of chromatography–mass spectrometry metabolomics data

Introduction

Data processing is one of the biggest problems in metabolomics, given the high number of samples analyzed and the need of multiple software packages for each step of the processing workflow.

Objectives

Merge in the same platform the steps required for metabolomics data processing.

Methods

KniMet is a workflow for the processing of mass spectrometry-metabolomics data based on the KNIME Analytics platform.

Results

The approach includes key steps to follow in metabolomics data processing: feature filtering, missing value imputation, normalization, batch correction and annotation.

Conclusion

KniMet provides the user with a local, modular and customizable workflow for the processing of both GC–MS and LC–MS open profiling data.

     

Pseudo current density maps of electrophysiological heart,nerve or brain function and their physical basis

Background

In recent years the visualization of biomagnetic measurement data by so-called pseudo current density maps or Hosaka-Cohen (HC) transformations became popular.

Methods

The physical basis of these intuitive maps is clarified by means of analytically solvable problems.

Results

Examples in magnetocardiography, magnetoencephalography and magnetoneurography demonstrate the usefulness of this method.

Conclusion

Hardware realizations of the HC-transformation and some similar transformations are discussed which could advantageously support cross-platform comparability of biomagnetic measurements.

     

Biodiversity impacts from water consumption on a global scale for use in life cycle assessment

Purpose

Agriculture is a major water user worldwide, potentially depriving many ecosystems of water. Comprehensive global impact assessment methodologies are therefore required to assess impacts from water consumption on biodiversity. Since scarcity of water, as well as species richness, varies greatly between different world regions, a spatially differentiated approach is needed. Therefore, our aim is to enhance a previously published methodology in terms of spatial and species coverage.

Methods

We developed characterization factors for lifecycle impact assessment (LCIA) targeting biodiversity loss of various animal taxa (i.e., birds, reptiles, mammals, and amphibians) in wetlands. Data was collected for more than 22,000 wetlands worldwide, distinguishing between surface water- and groundwater-fed wetlands. Additionally, we account for a loss of vascular plant species in terrestrial ecosystems, based on precipitation. The characterization factors are expressed as global fractions of potential species extinctions (PDF) per cubic meter of water consumed annually and are developed with a spatial resolution of 0.05 arc degrees. Based on the geographic range of species, as well as their current threat level, as indicated by the International Union for Conservation of Nature (IUCN), we developed a vulnerability indicator that is included in the characterization factor.

Results and discussion

Characterization factors have maximal values in the order of magnitude of 10^?11 PDF·year/m³ for animal taxa combined and 10^?12 PDF·year/m³ for vascular plants. The application of the developed factors for global cultivation of wheat, maize, cotton, and rice highlights that the amount of water consumption alone is not sufficient to indicate the places of largest impacts but that species richness and vulnerability of species are indeed important factors to consider. Largest impacts are calculated for vascular plants in Madagascar, for maize, and for animal taxa; in Australia and the USA for surface water consumption (cotton); and in Algeria and Tunisia for groundwater consumption (cotton).

Conclusions

We developed a spatially differentiated approach to account for impacts from water consumption on a global level. We demonstrated its functionality with an application to a global case study of four different crops.

     

Analysis of influencing factor of coexisting prediabetes and prehypertension in adult residents of Jilin Province

Background

To explore the risk factors of coexisting prediabetes and prehypertension, to provide theoretical basis for early intervention.

Methods

A multi-stage stratified random cluster sampling method was used to randomly select adult residents from Jilin Province in 2013 for questionnaire surveys, physical examinations, and laboratory tests.

Results

The prevalence of coexisting prediabetes and prehypertension in Jilin Province was 11.3%. The binary Logistic regression results showed that age, sex, education, triglyceride (TG), BMI, waist circumference and alcohol consumption were the effects of factor coexisting prediabetes and prehypertension.

Conclusion

It is important to pay attention to the early stage of hypertension and diabetes, control the transition from prehypertension and prediabetes to hypertension and diabetes, and improve the health of residents.

     

What stabilizes biological soil crusts in the Negev Desert?

Aims

Biological soil crusts (biocrusts) are widespread in many drylands, where plant growth is limited due to water scarcity. One of their most important functions is the stabilization of the topsoil, particularly in regions with sandy soils prone to desertification. Since the mechanisms playing a role in soil stabilization are poorly understood, this study aims to shed light on the connection between crust stability and different cementing agents.

Methods

We measured the penetration resistance and the concentrations of different cementing agents of biocrusts in the Israeli Negev Desert. Structural equation modelling was performed to examine the direct and indirect effects of the variables analyzed and identify variables that are best able to explain the observed patterns of penetration resistance.

Results

All observed variables showed a high variability within and between sites. Structural equation modelling revealed that the main parameters explaining penetration resistance are the content of fines and the electrical conductivity, while carbonates and organic carbon only have an indirect effect.

Conclusions

Our results suggest that adding silt and clay to (natural or induced) biocrusts is very likely to produce stronger, more stable crusts, which will be more effective in combating desertification and improve their ability to survive trampling by livestock.

     

Metabolomic profiling of maternal hair suggests rapid development of intrahepatic cholestasis of pregnancy

Introduction

Intrahepatic cholestasis of pregnancy (ICP) is a common maternal liver disease; development can result in devastating consequences, including sudden fetal death and stillbirth. Currently, recognition of ICP only occurs following onset of clinical symptoms.

Objective

Investigate the maternal hair metabolome for predictive biomarkers of ICP.

Methods

The maternal hair metabolome (gestational age of sampling between 17 and 41 weeks) of 38 Chinese women with ICP and 46 pregnant controls was analysed using gas chromatography–mass spectrometry.

Results

Of 105 metabolites detected in hair, none were significantly associated with ICP.

Conclusion

Hair samples represent accumulative environmental exposure over time. Samples collected at the onset of ICP did not reveal any metabolic shifts, suggesting rapid development of the disease.

     

Quantitative analysis of tetrahydrofolate metabolites from <Emphasis Type="Italic">clostridium autoethanogenum</Emphasis>

Introduction

Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.

Objective

To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.

Methods

Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.

Results

Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.

Conclusion

Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

     

Extraction of natural products from bark of <Emphasis Type="Italic">Betula pendula</Emphasis> using ionic liquids

Introduction

Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.

Objectives

To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.

Methods

The extracted compounds were analyzed by LC/MS and GC/MS.

Results

The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.

Conclusion

From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.