Ventilation rates and health: multidisciplinary review of the scientific literature.

UNLABELLED: The scientific literature through 2005 on the effects of ventilation rates on health in indoor environments has been reviewed by a multidisciplinary group. The group judged 27 papers published in peer-reviewed scientific journals as providing sufficient information on both ventilation rates and health effects to inform the relationship. Consistency was found across multiple investigations and different epidemiologic designs for different populations. Multiple health endpoints show similar relationships with ventilation rate. There is biological plausibility for an association of health outcomes with ventilation rates, although the literature does not provide clear evidence on particular agent(s) for the effects. Higher ventilation rates in offices, up to about 25 l/s per person, are associated with reduced prevalence of sick building syndrome (SBS) symptoms. The limited available data suggest that inflammation, respiratory infections, asthma symptoms and short-term sick leave increase with lower ventilation rates. Home ventilation rates above 0.5 air changes per hour (h(-1)) have been associated with a reduced risk of allergic manifestations among children in a Nordic climate. The need remains for more studies of the relationship between ventilation rates and health, especially in diverse climates, in locations with polluted outdoor air and in buildings other than offices. PRACTICAL IMPLICATIONS: Ventilation with outdoor air plays an important role influencing human exposures to indoor pollutants. This review and assessment indicates that increasing ventilation rates above currently adopted standards and guidelines should result in reduced prevalence of negative health outcomes. Building operators and designers should avoid low ventilation rates unless alternative effective measures, such as source control or air cleaning, are employed to limit indoor pollutant levels.

Olfactory detection of ozone and D-limonene: reactants in indoor spaces.

UNLABELLED: Young adult subjects who occupied a well-ventilated space with low background of level of ozone achieved via carbon-filtration could detect ozone odor at 7 ppb, lower than expected from archival compilations. The outcome was not inconsistent, however, with some observations of recognition, beyond mere detection, at about 15-20 ppb. Individual differences in sensitivity lay at or just below an order of magnitude, rare in olfactory testing and indicative of precision. In a study of d-limonene, subjects again showed high sensitivity and small individual differences. The subjects could detect the odor at 8 and 15 ppb, depending upon whether they occupied a space with or without carbon filtration, respectively. The results argue for use of carbon filtration to measure sensitivity most stringently, although absence of filtration seems not to incur a large penalty. The protocol used here, with collection of hundreds of judgments in a day, yet with little net exposure of the subject to odorant; with verifiably stable delivery; and with analytical confirmation of level should reduce tolerance for outcomes of large differences among subjects and among studies. PRACTICAL IMPLICATIONS: Humans manifested much higher sensitivity to ozone and D-limonene than commonly thought, a pattern revealing itself more broadly in olfactory studies as testing improves and analytical confirmation of delivery becomes more common. Published databases, with errors of +/-1000%, often badly underestimate sensitivity and can thereby encourage use of higher concentrations of compounds, particularly VOCs, than relevant in studies of reactive indoor chemistry.

Cutoff in detection of eye irritation from vapors of homologous carboxylic acids and aliphatic aldehydes.

Using neat vapors of selected homologous aldehydes (decanal, undecanal, dodecanal) and carboxylic acids (pentanoic, hexanoic, heptanoic, octanoic, nonanoic), we explored the point where a certain homolog (and all larger ones) becomes undetectable by eye irritation (i.e. by ocular chemesthesis). This phenomenon has been observed in other homologous series that also reach a break-point, or cutoff, in chemesthetic detection. Participants (11

Odor and chemesthesis from brief exposures to TXIB.

UNLABELLED: An experiment explored ability of subjects to detect vapors of the plasticizer TXIB (2,2,4-trimethyl-1,3-pentanediol diisobutyrate) and ethanol via olfaction and via ocular and nasal chemesthesis, i.e. chemically stimulated feel. Testing, tailored to the sensitivity of each subject, produced psychometric functions for individuals. Olfactory detection of TXIB began at concentrations below 1 ppb (v/v), with 50% correct detection at 1.2 ppb. (Comparable detection for ethanol occurred almost two orders of magnitude higher.) Chemesthetic detection of TXIB began at about 500 ppb, with 50% correct detection at 2.1 ppm for the eye and 4.6 ppm for the nose, both close to saturated vapor concentration. (Comparable detection for ethanol occurred essentially three orders of magnitude higher.) Suggestions that TXIB plays a role in generation of irritative symptoms at concentrations in the range of parts-per-billion need to reckon with a conservatively estimated 200-fold gap between the levels putatively 'responsible' for the symptoms and those even minimally detectable via chemesthesis. Neither the variable of exposure duration nor that of mixing offers a likely explanation. Inclusion of ethanol in the study allowed comparisons pertinent to issues of variability in human chemoreception. An interpretation of the psychometric functions for individuals across materials and perceptual continua led to the conclusion that use of concentration as the metric of detection in olfaction inflates individual differences. PRACTICAL IMPLICATIONS: This study indicated that the plasticizer TXIB could contribute odor at concentrations in the range of parts-per-billion, but could hardly contribute sensory irritation per se, as alleged in reports of some field studies where TXIB has existed amongst many other organic compounds.

Reduced hospitalization cost for patients with pectus excavatum treated using minimally invasive surgery.

BACKGROUND: Currently, few data exist regarding the relative costs associated with open and minimally invasive pectus excavatum repair. The aim of this study was to compare the surgical and hospitalization costs for these two surgical techniques and to identify factors responsible for cost differences. METHODS: A retrospective review of hospital charts, patient and parent questionnaires, and hospital accounting records was performed for 68 patients who underwent surgical correction of pectus excavatum between June 1996 and December 1999. RESULTS: In this series, 25 patients underwent open repair, whereas 43 patients underwent minimally invasive repair of pectus excavatum (MIRPE). The patient ages ranged from 4 to 19 years. The average ages for open repair (12 years) and MIRPE (11 years) did not differ significantly. As compared with open repair, MIRPE was associated with a 27% lower overall cost of hospitalization ( p < 0.05). The operating room costs were 12% higher for the patients who underwent MIRPE ( p < 0.05). The mean operative time for open repair was 3 h 15 min, whereas MIRPE required 1 h 10 min ( p < 0.001). The hospital stay for open repair averaged 4.4 days, as compared with 2.4 days for MIRPE ( p < 0.001). In contrast to other published series, the postoperative analgesia after MIRPE in this series consisted of narcotics, ketorolac, and methocarbamol. No patient received epidural analgesia, regardless of the repair technique selected. The postoperative complication rate was 4% in the open group and 14% in the MIRPE group. Most of the patients treated with either open or MIRPE reported postoperative oral narcotic usage for 2 weeks or less and returned to routine activities within 3 weeks. The patients and parents alike reported good to excellent overall outcomes in 85% or more of the open repair cases and 90% or more of the MIRPE cases. CONCLUSIONS: These data demonstrate for the first time that the use of an alternate pain management strategy including, narcotics, NSAIDs, and methocarbamol, but without epidural catheters, results in reduced hospital length of stay and decreased overall hospitalization costs for MIRPE, as compared with open pectus repair. This cost benefit was achieved without compromising pain management or patient satisfaction with surgical care.

Orbital response indicates nasal pungency: analysis of biomechanical strain on the skin.

Stimulation of the human nasal passage with pungent vapor elicits motor responses in a zone around the eye. This investigation addressed whether quantification of such responses, particularly activity of the orbicularis oculi muscle, could yield a sensitive index of nasal pungency. We placed an array of small, high-contrast targets just beneath the lower eyelid and videotaped their movement to capture deformation of the skin atop the orbicularis oculi during 3 s stimulation with pungent concentrations of ethyl acetate. Eleven subjects participated. Analysis of the movements served to determine mechanical strain, which yielded a single index that we termed 'maximum strain'. This increased with concentration of the vapor and with time during and just after stimulation. Comparison with psychophysical data showed that the strain became evident at concentrations just detectable as pungent. Maximum strain measured on the skin shows promise as an objective index of pungency.

Human breathing and eye blink rate responses to airborne chemicals.

Increased levels of air pollution have been linked with morbidity and mortality, but mechanisms linking physiologic responses to quality of life and productivity issues remain largely unknown. Individuals often report irritation of the nose and/or eyes upon exposures to environmental contaminants. Evaluation of these self-reports would be greatly aided by the development of valid physiological markers. Chamber studies (unencumbered exposures) of nonsmoker responses to environmental tobacco smoke offer two candidate end points: (a) Tidal volume increases and breathing frequency declines with stimuli that elicit only moderate irritation. (b) Eye blink rate increases only with a concentration sufficiently high to cause progressive worsening of eye irritation with prolonged exposure. Experiments with very brief nasal-only presentations also suggest the value of breathing changes as sensitive markers of irritation: (a) Tidal volume is inversely related to perceived nasal irritation (NI) intensity in both normal and anosmic (lacking olfactory input) individuals, although normals exhibit greater NI sensitivity. (b) Inhalation duration, in both groups, declines only with trigeminal activation sufficient to cause readily perceptible NI in anosmics. Changes in eye blink rate and breathing may be useful in the investigation of irritation and other effects of air pollution, and could be quite useful in investigations of mixtures of volatile organic compounds.

Ocular and nasal trigeminal detection of butyl acetate and toluene presented singly and in mixtures.

To probe into the rules of trigeminal chemosensory agonism in a binary mixture of chemicals we measured, first, the detectability (i.e., psychometric) function for eye irritation and for nasal pungency of butyl acetate and toluene, singly. (To avoid olfactory biases, nasal pungency was measured in a group of anosmics, i.e., persons lacking a functional sense of smell.) Then, based on the detectability function obtained for the individual chemicals, we prepared mixtures where the 2 components varied in their relative proportions but, if a simple rule of complete sensory agonism (in the sense of dose-additivity) were to hold, the mixtures should be as detectable as the reference concentration of each of the single chemicals. For both trigeminal endpoints (i.e., eye irritation and nasal pungency), the results showed that stimuli of relatively low detectability did show complete sensory agonism, whereas stimuli of relatively high detectability fell short of complete sensory agonism when compared with the detectability of the single substances. Further testing of additional binary and higher order mixtures will confirm whether or not a structure-activity model of trigeminal chemosensory impact of single chemicals, based on selected physicochemical parameters of the stimuli, can also be applied to chemical mixtures.

Psychometrics of odor quality discrimination: method for threshold determination.

There is no natural physical continuum for odor quality along which an odor quality discrimination (OQD) threshold can be measured. In an attempt to overcome this problem, the substitution-reciprocity (SURE) method defines a framework for the measurement of an OQD threshold. More specifically, it (i) defines a threshold concept for OQD, including the quantification of qualitative change of the stimulus, and (ii) suggests how to avoid perceived intensity as an unwanted cue for discrimination. In doing this, the psychometric properties of odor quality in the case of eugenol and citral are investigated using both discrimination (experiment 1) and scaling (experiment 2). Based on discriminatory responses, a change of approximately one-third in stimulus content was needed to reach the OQD threshold for eugenol and citral.

Connection between chromatographic data and biological data.

There are no previous references to the direct use of GLC data in the correlation of biological processes, but we show that GLC retention data can be used in the correlation of several such processes involving gaseous solutes. There are a number of reports of RP-HPLC and MEKC data being used in the correlation of biological processes, but they are mostly restricted as to the number and type of solute studied. We show that if chromatographic data are used to obtain solvation descriptors for solutes, and if these descriptors are then used in the correlation of biological processes, that this indirect connection is a much more powerful and generally applicable method than is the direct connection between chromatographic data and biological data.

Quantification of odor quality.

The relationship between odor quality and molecular properties is arguably the most important issue in olfaction. Despite sophistication in the chemical characterization of molecules, accompanying perceptual characterization has had little quantitative usefulness, relying mostly on enumerative description. As a result of weak interest in the topic outside industry and little agreement regarding how to measure quality, the field of olfactory psychophysics has failed to develop a substantial database for odor quality and has offered little help to other researchers, e.g. neurobiologists, in choice of stimuli, interpretation of outcome or testable hypotheses. This review scrutinizes how psychophysicists and others have measured quality and offers criteria for useful techniques. Most measures have had a subjective component that makes them anachronistic with modern methodology in experimental behavioral science, indeterminate regarding the extent of individual differences, unusable with infrahumans and of unproved ability to discern small differences. Techniques based upon performance, rather than on the more common reporting of mental content, offer firmer possibilities for growth. These techniques inevitably tap the discriminative basis of perception. The nonsubjective techniques have high sensitivity, can have counterparts in infrahuman research, are suitable to examine individual differences and yield non-negotiable answers with potential archival value. Discriminative techniques have their limitations, too-principally excess sensitivity that abridges their use to comparisons between similar-smelling stimuli. Research has begun to extend that range and may overcome the limitation. Application of discriminative methods may have the side-effect of shifting focus in structure-activity research from searches for molecular least common denominators that underlie often vague similarity to the search for molecular properties of importance in discrimination of small differences.

Latency and accuracy of discriminations of odor quality between binary mixtures and their components.

Subjects made timed, same-different discriminations of odor quality, with the following principal findings: (i) latency reflected accuracy, with difficult discriminations, i.e. those between 50-50 mixtures and their components, requiring more time than less difficult discriminations, i.e. those between unmixed chemicals. This finding demonstrated the face validity of latency as a measure of qualitative similarity. (ii) Latency provided better resolution among pairs of odors than did errors of discrimination. This finding demonstrated the utility of collecting response times. (iii) Latency-based similarities among odors tested previously predicted similarities among pairs not yet tested. This finding demonstrated internal/predictive validity. (iv) A signal detection model assuming a differencing strategy best described the pattern of errors. Subjects appeared to make relative judgements regarding quality. (v) Finally, latency-based similarities between mixtures and their components demonstrated additivity. This finding suggested that binary mixtures fall on straight lines connecting their components in 'odor-space'.

Comparison of two stimulus-delivery systems for measurement of nasal pungency thresholds.

Using representative members of each of three homologous series of chemicals-ketones, acetates and alcohols-we measured nasal pungency thresholds in anosmics via two stimulus-delivery systems. The first system consists of the fairly commonly used 270 ml, plastic 'squeeze bottles'. The second system consists of 1900 ml, glass vessels with Teflon tubing and nose-pieces. Although bulkier and more susceptible to mechanical breakage, the glass vessels possess advantages that can allow them to provide 'environmentally realistic' chemosensory thresholds, i.e. thresholds closer in absolute values to those that might be obtained under whole-body exposures. Such advantages include a larger volume of the vapor-source to accommodate whole sniffs, and a tight nose-nose-piece connection to avoid stimulus dilution. The outcome revealed that, for every chemical, the glass vessels provided nasal pungency thresholds significantly lower than those provided by the squeeze bottles. The difference amounted, on average, to a factor of 4.6, though the relative potency of the compounds remained the same under both systems. Additionally, when tested with the highest homologues used here, namely, octyl acetate and 1-octanol, anosmics using the glass vessels had little or no difficulty achieving the criterion for threshold whereas they did have difficulty when using the squeeze bottles.

Measurement of sensitivity to olfactory flavor: application in a study of aging and dentures.

Olfaction involves a dual sensory process for perceiving odors orthonasally (through the nostrils) and retronasally (through the mouth). This investigation entailed developing a measure of sensitivity to an odor delivered in an orally sampled food (orange flavoring in a sucrose-sweetened gelatin) and examining sensitivity in the elderly. In experiment 1, olfactory flavor sensitivity was 49 times lower in elderly (n = 21) than in young (n = 28) subjects. In experiment 2, with 73 elderly women, higher olfactory flavor sensitivity correlated significantly with higher orthonasal perception (Connecticut Chemosensory Clinical Research Center test). Some women, however, exhibited low olfactory flavor sensitivity despite high orthonasal perception; none had high olfactory flavor sensitivity and low orthonasal perception. Those who wore complete or palatal covering dentures had lower olfactory flavor sensitivity than those who were dentate or wore dentures that did not cover the palate. Through multiple regression analysis, orthonasal perception and denture status were found to be independent contributors to predicting olfactory flavor sensitivity. In summary, elderly subjects showed depressed olfactory flavor sensitivity (i.e. retronasal sensitivity) that related to poor orthonasal olfactory perception and denture characteristic. Thus, while good orthonasal olfaction may be necessary for good olfactory flavor sensitivity, it is not sufficient. Other factors, some associated with oral conditions, may impede release and retronasal transport of odors from the mouth to the olfactory receptors.

Chemosensory detectability of 1-butanol and 2-heptanone singly and in binary mixtures.

Using 1-butanol and 2-heptanone as stimuli, we measured detectability (i.e., psychometric) functions for the odor, nasal pungency, and eye irritation of these two substances alone and in binary mixtures. Nasal pungency responses were tested in subjects lacking olfaction (i.e., anosmics) for whom odors do not interfere. Eye irritation responses were tested in normosmics and anosmics, and found to be similar in both groups so their results were pooled. When all stimuli--single and mixtures--were transformed into concentration units of one (or the other) chemical, a single function could fit all data from the same sensory end point with a correlation coefficient of 0.91 or higher. The outcome lends support, as a first approximation, to the notion of chemosensory agonism, in the sense of dose additivity, between the members of binary mixtures presented at perithreshold levels.

Gastrointestinal autonomic nerve tumors in the pediatric population: a report of four cases and a review of the literature.

BACKGROUND: Gastrointestinal autonomic nerve tumors (GANTs) are a subpopulation of gastrointestinal stromal tumors (GISTs) that are characterized by ultrastructural features resembling enteric autonomic nerve cells, without epithelial, Schwannian, or smooth muscle differentiation. Delineation of the clinicopathologic features of GANT in the pediatric population is lacking. METHODS: The clinicopathologic and outcome data for four pediatric patients with GANT are presented. The data from these patients and four previously reported pediatric patients are summarized and compared with data for GANT in adults. RESULTS: All four cases occurred in females at a mean age of 12.5 years. The primary tumor site was the stomach in all cases, and the mean tumor size was 6.3 cm. Immunocytochemical and ultrastructural examination were essential in distinguishing GANT from GIST in all cases by identifying features of neural origin (neuron specific enolase in all four cases, NFP in three cases, S-100 in two cases, dense core neurosecretory granules in all four cases, and neuritelike processes in all four cases), while failing to identify features of myogenic origin (no desmin, smooth muscle actin, myofilaments, or dense bodies were found in any of the cases). Primary treatment was surgical, with chemotherapy administered to 1 patient at the time of recurrence. All patients are alive after a mean follow-up of 60 months (range, 8 months to 9 years). CONCLUSIONS: Similarities of pediatric GANT to GANT in adults include the need for a high index of suspicion for diagnosis; comparable histopathologic, immunohistochemical, and ultrastructural features; and surgery as the primary therapy. Distinguishing features in children may be a prevalence among females in the second decade, a predominance of smaller gastric tumors, and a positive prognostic value of younger age.

Odor identification: perceptual and semantic dimensions.

Five studies explored identification of odors as an aspect of semantic memory. All dealt in one way or another with the accessibility of acquired olfactory information. The first study examined stability and showed that, consistent with personal reports, people can fail to identify an odor one day yet succeed another. Failure turned more commonly to success than vice versa, and once success occurred it tended to recur. Confidence ratings implied that subjects generally knew the quality of their answers. Even incorrect names, though, often carried considerable information which sometimes reflected a semantic and sometimes a perceptual source of errors. The second study showed that profiling odors via the American Society of Testing and Materials list of attributes, an exercise in depth of processing, effected no increment in the identifiability/accessibility beyond an unelaborated second attempt at retrieval. The third study showed that subjects had only a weak ability to predict the relative recognizability of odors they had failed to identify. Whereas the strength of the feeling that they would 'know' an answer if offered choices did not associate significantly with performance for odors, it did for trivia questions. The fourth study demonstrated an association between ability to discriminate among one set of odors and to identify another, but this emerged only after subjects had received feedback about identity, which essentially changed the task to one of recognition and effectively stabilized access. The fifth study illustrated that feedback improves performance dramatically only for odors involved with it, but that mere retrieval leads to some improvement. The studies suggest a research agenda that could include supplemental use of confidence judgments both retrospectively and prospectively in the same subjects to indicate the amount of accessible semantic information; use of second and third guesses to examine subjects' simultaneously held hypotheses about identity; use of category cuing or similar techniques to discover the minimum semantic information needed to precipitate identification; some use of subjects trained in quantitative descriptive analysis to explore whether such training enhances semantic memory; and judicious use of mixtures to explore perceptual versus semantic errors of identification.

Trigeminal and olfactory chemosensory impact of selected terpenes.

In Experiment 1, four normosmics and four anosmics (three congenital, one idiopathic) provided odor and nasal pungency thresholds, respectively, for the following terpenes: delta3-carene, p-cymene, linalool, 1.8-cineole, and geraniol, plus the structurally related compound cumene. Additionally, all subjects provided nasal localization (i.e., right/left) and eye irritation thresholis. Trigeminally mediated thresholds (i.e., nasal pungency, nasal localization, and eye irritation) lay about three orders of magnitude above odor thresholds, which ranged between 0.1 and 1.7 ppm. The results implied uniform chemesthetic sensitivity across tasks and sites of impact. In Experiment 2, normosmics and anosmics provided odor and nasal pungency thresholds, respectively, for three pairs of isomeric terpenes: alpha- and gamma-terpinene, alpha- and beta-pinene, and R(+)- and S(-)-limonene. Odor thresholds ranged between 1.4 and 19 ppm, that is, about an order of magnitude higher than those of the previous terpenes, with no substantial differences between odor thresholds of members of a pair. Regarding chemesthetic impact, only alpha-terpinene evoked nasal pungency. The overall outcome suggests comparable trigeminal chemosensitivity between nose and eyes and between normosmics and anosmics, as shown before for homologous n-alcohols. It also lends support to a previously derived solvation model of the chemesthetic potency of airborne substances, and indicates the likely importance of certain molecular-size restrictions for effective trigeminal impact.

Nasal pungency and odor of homologous aldehydes and carboxylic acids.

Airborne substances can stimulate both the olfactory and the trigeminal nerve in the nose, giving rise to odor and pungent (irritant) sensations, respectively. Nose, eye, and throat irritation constitute common adverse effects in indoor environments. We measured odor and nasal pungency thresholds for homologous aliphatic aldehydes (butanal through octanal) and carboxylic acids (formic, acetic, butanoic, hexanoic, and octanoic). Nasal pungency was measured in subjects lacking olfaction (i.e., anosmics) to avoid odor biases. Similar to other homologous series, odor and pungency thresholds declined (i.e., sensory potency increased) with increasing carbon chain length. A previously derived quantitative structure-activity relationship (QSAR) based on solvation energies predicted all nasal pungency thresholds, except for acetic acid, implying that a key step in the mechanism for threshold pungency involves transfer of the inhaled substance from the vapor phase to the receptive biological phase. In contrast, acetic acid - with a pungency threshold lower than predicted - is likely to produce threshold pungency through direct chemical reaction with the mucosa. Both in the series studied here and in those studied previously, we reach a member at longer chain-lengths beyond which pungency fades. The evidence suggests a biological cut-off, presumably based upon molecular size, across the various series.

Trigeminal and olfactory sensitivity: comparison of modalities and methods of measurement.

OBJECTIVE: The principal objective was to chart sensitivity for human nasal irritation by alternative psychophysical methods, namely, a common detection procedure versus a nasal lateralization procedure that required the subject to indicate whether a vapor had stimulated the left or right nostril. This objective relates to the broader issues as to (a) whether subjects with normal olfaction (normosmics) can yield, through novel methodology, an index of sensitivity to nasal irritation comparable with that obtained from subjects without olfaction (anosmics) and (b) whether both types of subjects have similar irritation sensitivity in general. This study sought to gauge interconvertability both between types of subjects and between modes of stimulus presentation for irritative and, where appropriate, olfactory stimulation. METHODS: Static dilution series of four n-aliphatic alcohols, chosen to represent volatile organic compounds (VOCs), provided the source of calibrated olfactory and irritative vapors emitted from their squeezable containers into the nose or eye either by a mechanical device or by hand. Standard psychophysical methodology (forced-choice; ascending strength of stimulation) served to chart detection thresholds for irritation and odor and an analogous procedure served to chart the threshold for localization of stimulation. RESULTS: Within the limits of resolution, detection thresholds and nasal localization thresholds yielded comparable indices of the potency of the VOCs to evoke nasal irritation. The thresholds agreed well with those for detection of eye irritation, though only the eyes proved to be capable of detecting irritation from l-octanol. The method of emitting the stimulus had little material effect on measures of either irritative or olfactory detection. CONCLUSIONS: The threshold for nasal localization offers a suitable way to measure nasal irritation in normosmic persons. Olfactory stimulation does not interfere with the measure since subjects cannot localize on that basis. Anosmic and normosmic persons have comparable sensitivity to nasal and ocular irritation. If anosmic persons have any lower sensitivity, as sometimes claimed, it would seem to have only trivial consequences for estimates of the irritative potency of VOCs.