Bioremediation of PAHs - Limitations and soultions

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

Controllo dell'atto di prensione: coinvolgimento dell'area V6A nell'orientamento del polso

Prehension in an act of coordinated reaching and grasping. The reaching component is concerned with bringing the hand to object to be grasped (transport phase); the grasping component refers to the shaping of the hand according to the object features (grasping phase) (Jeannerod, 1981). Reaching and grasping involve different muscles, proximal and distal muscles respectively, and are controlled by different parietofrontal circuit (Jeannerod et al., 1995): a medial circuit, involving area of superior parietal lobule and dorsal premotor area 6 (PMd) (dorsomedial visual stream), is mainly concerned with reaching; a lateral circuit, involving the inferior parietal lobule and ventral premotor area 6 (PMv) (dorsolateral visual stream), with grasping. Area V6A is located in the caudalmost part of the superior parietal lobule, so it belongs to the dorsomedial visual stream; it contains neurons sensitive to visual stimuli (Galletti et al. 1993, 1996, 1999) as well as cells sensitive to the direction of gaze (Galletti et al. 1995) and cells showing saccade-related activity (Nakamura et al. 1999; Kutz et al. 2003). Area V6A contains also arm-reaching neurons likely involved in the control of the direction of the arm during movements towards objects in the peripersonal space (Galletti et al. 1997; Fattori et al. 2001). The present results confirm this finding and demonstrate that during the reach-to-grasp the V6A neurons are also modulated by the orientation of the wrist. Experiments were approved by the Bioethical Committee of the University of Bologna and were performed in accordance with National laws on care and use of laboratory animals and with the European Communities Council Directive of 24th November 1986 (86/609/EEC), recently revised by the Council of Europe guidelines (Appendix A of Convention ETS 123). Experiments were performed in two awake Macaca fascicularis. Each monkey was trained to sit in a primate chair with the head restrained to perform reaching and grasping arm movements in complete darkness while gazing a small fixation point. The object to be grasped was a handle that could have different orientation. We recorded neural activity from 163 neurons of the anterior parietal sulcus; 116/163 (71%) neurons were modulated by the reach-to-grasp task during the execution of the forward movements toward the target (epoch MOV), 111/163 (68%) during the pulling of the handle (epoch HOLD) and 102/163 during the execution of backward movements (epoch M2) (t_test, p ≤ 0.05). About the 45% of the tested cells turned out to be sensitive to the orientation of the handle (one way ANOVA, p ≤ 0.05). To study how the distal components of the movement, such as the hand preshaping during the reaching of the handle, could influence the neuronal discharge, we compared the neuronal activity during the reaching movements towards the same spatial location in reach-to-point and reach-to-grasp tasks. Both tasks required proximal arm movements; only the reach-to-grasp task required distal movements to orient the wrist and to shape the hand to grasp the handle. The 56% of V6A cells showed significant differences in the neural discharge (one way ANOVA, p ≤ 0.05) between the reach-to-point and the reach-to-grasp tasks during MOV, 54% during HOLD and 52% during M2. These data show that reaching and grasping are processed by the same population of neurons, providing evidence that the coordination of reaching and grasping takes place much earlier than previously thought, i.e., in the parieto-occipital cortex. The data here reported are in agreement with results of lesions to the medial posterior parietal cortex in both monkeys and humans, and with recent imaging data in humans, all of them indicating a functional coupling in the control of reaching and grasping by the medial parietofrontal circuit.

Geochemistry, petrogenesis and tectonic setting of ophiolites and mafic-ultramafic complexes in the Northeastern Aegean Region

In this study two ophiolites and a mafic-ultramafic complexes of the northeastern Aegean Sea, Greece, have been investigated to re-evaluate their petrogenetic evolution and tectonic setting. These complexes are: the mafic-ultramafic complex of Lesvos Island and the ophiolites of Samothraki Island and the Evros area. In order to examine these complexes in detail whole-rock major- and trace-elements as well as Sr and Nd isotopes, and minerals were analysed and U-Pb SHRIMP ages on zircons were determined. The mafic-ultramafic complex of Lesvos Island consists of mantle peridotite thrusted over a tectonic mélange containing metasediments, metabasalts and a few metagabbros. This succession had previously been interpreted as an ophiolite of Late Jurassic age. The new field and geochemical data allow a reinterpretation of this complex as representing an incipient continental rift setting that led to the subsequent formation of the Meliata-Maliac-Vardar branches of Neotethys in Upper Permian times (253 ± 6 Ma) and the term “Lesvos ophiolite” should be abandoned. With proceeding subduction and closure of the Maliac Ocean in Late Jurassic times (155 Ma) the Lesvos mafic-ultramafic complex was obducted. Zircon ages of 777, 539 and 338 Ma from a gabbro strongly suggest inheritance from the intruded basement and correspond to ages of distinct terranes recently recognized in the Hellenides (e.g. Florina terrane). Geochemical similar complexes which contain rift associations with Permo-Triassic ages can be found elsewhere in Greece and Turkey, namely the Teke Dere Thrust Sheet below the Lycian Nappes (SW Turkey), the Pindos subophiolitic mélange (W Greece), the Volcanosedimentary Complex on Central Evia Island (Greece) and the Karakaya Complex (NW Turkey). This infers that the rift-related rocks from Lesvos belong to an important Permo-Triassic rifting episode in the eastern Mediterranean. The ‘in-situ’ ophiolite of Samothraki Island comprises gabbros, sparse dykes and basalt flows as well as pillows cut by late dolerite dykes and had conventionally been interpreted as having formed in an ensialic back-arc basin. The results of this study revealed that none of the basalts and dolerites resemble mid-ocean ridge or back-arc basin basalts thus suggesting that the Samothraki ophiolite cannot represent mature back-arc basin crust. The age of the complex is regarded to be 160 ± 5 Ma (i.e. Oxfordian; early Upper Jurassic), which precludes any correlation with the Lesvos mafic-ultramafic complex further south (253 ± 6 Ma; Upper Permian). Restoration of the block configuration in NE Greece, before extensional collapse of the Hellenic hinterland and exhumation of the Rhodope Metamorphic Core Complex (mid-Eocene to mid-Miocene), results in a continuous ophiolite belt from Guevgueli in the NW to Samothraki in the SE, thus assigning the latter to the Innermost Hellenic Ophiolite Belt. In view of the data of this study, the Samothraki ophiolite represents a rift propagation of the Sithonia ophiolite spreading ridge into the Chortiatis calc-alkaline arc. The ophiolite of the Evros area consists of a plutonic sequence comprising cumulate and non-cumulate gabbros with plagiogranite veins, and an extrusive sequence of basalt dykes, massive and pillow lavas as well as pyroclastic rocks. Furthermore, in the Rhodope Massif tectonic lenses of harzburgites and dunites can be found. All rocks are spatially separated. The analytical results of this study revealed an intra-oceanic island arc setting for the Evros ophiolitic rocks. During late Middle Jurassic times (169 ± 2 Ma) an intra-oceanic arc has developed above a northwards directed intra-oceanic subduction zone of the Vardar Ocean in front of the Rhodope Massif. The boninitic, island arc tholeiitic and calc-alkaline rocks reflect the evolution of the Evros island arc. The obduction of the ophiolitic rocks onto the Rhodope basement margin took place during closure of the Vardar ocean basins. The harzburgites and dunites of the Rhodope Massif are strongly depleted and resemble harzburgites from recent oceanic island arcs. After melt extraction they underwent enrichment processes by percolating melts and fluids from the subducted slab. The relationship of the peridotites and the Evros ophiolite is still ambiguous, but the stratigraphic positions of the peridotites and the ophiolitic rocks indicate separated origin. The harzburgites and dunites most probably represent remnants of the mantle wedge of the island arc of the Rhodope terrane formed above subducted slab of the Nestos Ocean in late Middle Jurassic times. During collision of the Thracia terrane with the Rhodope terrane thrusting of the Rhodope terrane onto the Thracia terrane took place, whereas the harzburgites and dunites were pushed between the two terranes now cropping out on top of the Thracia terrane of the Rhodope Massif.

Excellent agreement between genetic and hydrogen breath tests for lactase deficiency and the role of extended symptom assessment

Clinical manifestations of lactase (LCT) deficiency include intestinal and extra-intestinal symptoms. Lactose hydrogen breath test (H2-BT) is considered the gold standard to evaluate LCT deficiency (LD). Recently, the single-nucleotide polymorphism C/T(-13910) has been associated with LD. The objectives of the present study were to evaluate the agreement between genetic testing of LCT C/T(-13910) and lactose H2-BT, and the diagnostic value of extended symptom assessment. Of the 201 patients included in the study, 194 (139 females; mean age 38, range 17-79 years, and 55 males, mean age 38, range 18-68 years) patients with clinical suspicion of LD underwent a 3-4 h H2-BT and genetic testing for LCT C/T(-13910). Patients rated five intestinal and four extra-intestinal symptoms during the H2-BT and then at home for the following 48 h. Declaring H2-BT as the gold standard, the CC(-13910) genotype had a sensitivity of 97% and a specificity of 95% with a of 0.9 in diagnosing LCT deficiency. Patients with LD had more intense intestinal symptoms 4 h following the lactose challenge included in the H2-BT. We found no difference in the intensity of extra-intestinal symptoms between patients with and without LD. Symptom assessment yielded differences for intestinal symptoms abdominal pain, bloating, borborygmi and diarrhoea between 120 min and 4 h after oral lactose challenge. Extra-intestinal symptoms (dizziness, headache and myalgia) and extension of symptom assessment up to 48 h did not consistently show different results. In conclusion, genetic testing has an excellent agreement with the standard lactose H2-BT, and it may replace breath testing for the diagnosis of LD. Extended symptom scores and assessment of extra-intestinal symptoms have limited diagnostic value in the evaluation of LD.

Liberation as Revolutionary Praxis: Rethinking Buddhism Materialism

While it is only in recent decades that scholars have begun to reconsider and problematize Buddhist conceptions of “freedom” and “agency,” the thought traditions of Asian Buddhism have for many centuries struggled with questions related to the issue of “liberation”—along with its fundamental ontological, epistemological and ethical implications. With the development of Marxist thought in the mid to late nineteenth century, a new paradigm for thinking about freedom in relation to history, identity and social change found its way to Asia, and confronted traditional religious interpretations of freedom as well as competing Western ones. In the past century, several attempts have been made—in India, southeast Asia, China and Japan—to bring together Marxist and Buddhist worldviews, with only moderate success (both at the level of theory and practice). This paper analyzes both the possibilities and problems of a “Buddhist materialism” constructed along Marxian lines, by focusing in particular on Buddhist and Marxist conceptions of “liberation.” By utilizing the theoretical work of Japanese “radical Buddhist” Seno’o Girō, I argue that the root of the tension lies with conceptions of selfhood and agency—but that, contrary to expectations, a strong case can be made for convergence between Buddhist and Marxian perspectives on these issues, as both traditions ultimately seek a resolution of existential determination in response to alienation. Along the way, I discuss the work of Marx, Engels, Gramsci, Lukàcs, Sartre, and Richard Rorty in relation to aspects of traditional (particularly East Asian Mahāyāna) Buddhist thought.

Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than in high oxygen cultures. Low oxygen during FGF2-mediated expansion resulted also in a significant increase in tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons as compared to high oxygen tension, but no corresponding effect was observed for dopamine release into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells.

Brain connectivity at different time-scales measured with EEG

We present an overview of different methods for decomposing a multichannel spontaneous electroencephalogram (EEG) into sets of temporal patterns and topographic distributions. All of the methods presented here consider the scalp electric field as the basic analysis entity in space. In time, the resolution of the methods is between milliseconds (time-domain analysis), subseconds (time- and frequency-domain analysis) and seconds (frequency-domain analysis). For any of these methods, we show that large parts of the data can be explained by a small number of topographic distributions. Physically, this implies that the brain regions that generated one of those topographies must have been active with a common phase. If several brain regions are producing EEG signals at the same time and frequency, they have a strong tendency to do this in a synchronized mode. This view is illustrated by several examples (including combined EEG and functional magnetic resonance imaging (fMRI)) and a selective review of the literature. The findings are discussed in terms of short-lasting binding between different brain regions through synchronized oscillations, which could constitute a mechanism to form transient, functional neurocognitive networks.

An Experience-Driven Pedagogy for the Instruction of Software Testing in Computer Science

In the realm of computer programming, the experience of writing a program is used to reinforce concepts and evaluate ability. This research uses three case studies to evaluate the introduction of testing through Kolb's Experiential Learning Model (ELM). We then analyze the impact of those testing experiences to determine methods for improving future courses. The first testing experience that students encounter are unit test reports in their early courses. This course demonstrates that automating and improving feedback can provide more ELM iterations. The JUnit Generation (JUG) tool also provided a positive experience for the instructor by reducing the overall workload. Later, undergraduate and graduate students have the opportunity to work together in a multi-role Human-Computer Interaction (HCI) course. The interactions use usability analysis techniques with graduate students as usability experts and undergraduate students as design engineers. Students get experience testing the user experience of their product prototypes using methods varying from heuristic analysis to user testing. From this course, we learned the importance of the instructors role in the ELM. As more roles were added to the HCI course, a desire arose to provide more complete, quality assured software. This inspired the addition of unit testing experiences to the course. However, we learned that significant preparations must be made to apply the ELM when students are resistant. The research presented through these courses was driven by the recognition of a need for testing in a Computer Science curriculum. Our understanding of the ELM suggests the need for student experience when being introduced to testing concepts. We learned that experiential learning, when appropriately implemented, can provide benefits to the Computer Science classroom. When examined together, these course-based research projects provided insight into building strong testing practices into a curriculum.

Variability of anti-PF4/heparin antibody results obtained by the rapid testing system ID-H/PF4-PaGIA

BACKGROUND: Recent studies have shown that a low clinical pretest probability may be adequate for excluding heparin-induced thrombocytopenia. However, for patients with intermediate or high pretest probability, laboratory testing is essential for confirming or refuting the diagnosis. Rapid assessment of anti-PF4/heparin-antibodies may assist clinical decision-making. OBJECTIVES: To evaluate the performance of rapid ID-H/PF4-PaGIA. In particular, we verified reproducibility of results between plasma and serum specimens, between fresh and frozen samples, and between different ID-H/PF4-polymer lots (polystyrene beads coated with heparin/PF4-complexes). PATIENTS/METHODS: The samples studied were 1376 plasma and 914 corresponding serum samples from patients investigated for suspected heparin-induced thrombocytopenia between January 2000 and October 2008. Anti-PF4/heparin-antibodies were assessed by ID-H/PF4-PaGIA, commercially available ELISAs and heparin-induced platelet aggregation test. RESULTS: Among 914 paired plasma/serum samples we noted discordant results (negative vs. low-titre positive) in nine instances (1%; 95%CI, 0.4-1.6%). Overall, agreement between titres assessed in plasma vs. serum was highly significant (Spearman correlation coefficient, 0.975; P < 0.0001). Forty-seven samples tested both fresh and after freezing/thawing showed a good agreement, with one discordant positive/negative result (Spearman correlation coefficient, 0.970; P < 0.0001). Among 1376 plasma samples we noted a strikingly variable incidence of false negative results (none - 82%; 95%CI, 66-98%), depending on the employed ID-H/PF4-polymer lot. Faulty lots can be recognized by titrating commercial positive controls and stored samples of HIT-patients. CONCLUSION: Laboratories performing the assay should implement stringent internal quality controls in order to recognize potentially faulty ID-H/PF4-polymer lots, thus avoiding false negative results.

Is low self-esteem a risk factor for depression? Findings from a longitudinal study of Mexican-origin youth

We examined the relation between low self-esteem and depression using longitudinal data from a sample of 674 Mexican-origin early adolescents who were assessed at age 10 and 12 years. Results supported the vulnerability model, which states that low self-esteem is a prospective risk factor for depression. Moreover, results suggested that the vulnerability effect of low self-esteem is driven, for the most part, by general evaluations of worth (i.e., global self-esteem), rather than by domain-specific evaluations of academic competence, physical appearance, and competence in peer relationships. The only domain-specific self-evaluation that showed a prospective effect on depression was honesty-trustworthiness. The vulnerability effect of low self-esteem held for male and female adolescents, for adolescents born in the United States versus Mexico, and across different levels of pubertal status. Finally, the vulnerability effect held when we controlled for several theoretically relevant 3rd variables (i.e., social support, maternal depression, stressful events, and relational victimization) and for interactive effects between self-esteem and the 3rd variables. The present study contributes to an emerging understanding of the link between self-esteem and depression and provides much needed data on the antecedents of depression in ethnic minority populations

Comparing growth trajectories of risk behaviors from late adolescence through young adulthood: An accelerated design

Risk behaviors such as substance use or deviance are often limited to the early stages of the life course. Whereas the onset of risk behavior is well studied, less is currently known about the decline and timing of cessation of risk behaviors of different domains during young adulthood. Prevalence and longitudinal developmental patterning of alcohol use, drinking to the point of drunkenness, smoking, cannabis use, deviance, and HIV-related sexual risk behavior were compared in a Swiss community sample (N = 2,843). Using a longitudinal cohort-sequential approach to link multiple assessments with 3 waves of data for each individual, the studied period spanned the ages of 16 to 29 years. Although smoking had a higher prevalence, both smoking and drinking up to the point of drunkenness followed an inverted U-shaped curve. Alcohol consumption was also best described by a quadratic model, though largely stable at a high level through the late 20s. Sexual risk behavior increased slowly from age 16 to age 22 and then remained largely stable. In contrast, cannabis use and deviance linearly declined from age 16 to age 29. Young men were at higher risk for all behaviors than were young women, but apart from deviance, patterning over time was similar for both sexes. Results about the timing of increase and decline as well as differences between risk behaviors may inform tailored prevention programs during the transition from late adolescence to adulthood.