The validated sun exposure questionnaire: association of objective and subjective measures of sun exposure in a Danish population-based sample.

BACKGROUND: Few questionnaires used in monitoring sun-related behaviour have been tested for validity. OBJECTIVES: We established the criteria validity of a questionnaire developed for monitoring population sun-related behaviour. METHODS: During May-August 2013, 664 Danes wore a personal electronic ultraviolet radiation (UVR) dosimeter for 1 week that measured their outdoor time and dose of erythemal UVR exposure. In the following week, they answered a questionnaire on their sun-related behaviour in the measurement week. RESULTS: Outdoor time measured by dosimetry correlated strongly with both outdoor time and the developed exposure scale measured in the questionnaire. Exposure measured in standard erythema dose (SED) by dosimetry correlated strongly with the exposure scale. In a linear regression model of UVR (SED) received, 41% of the variation was explained by skin type, age, week of participation and exposure scale, with exposure scale as the main contributor. The weekly sunburn fraction correlated strongly with the number of ambient sun hours (r = 0·73, P < 0·001). CONCLUSIONS: This criteria-validated questionnaire provides evidence of the exposure that the questionnaire aimed to measure. The evidence provided showed a strong link between the objectively measured behaviour and the behaviour measured by this survey construct. The questionnaire is the first validated tool to measure the UVR exposure in a national population-based sample.

Associations between bacterial communities of house dust and infant gut.

The human gut is host to a diverse and abundant community of bacteria that influence health and disease susceptibility. This community develops in infancy, and its composition is strongly influenced by environmental factors, notably perinatal anthropogenic exposures such as delivery mode (Cesarean vs. vaginal) and feeding method (breast vs. formula); however, the built environment as a possible source of exposure has not been considered. Here we report on a preliminary investigation of the associations between bacteria in house dust and the nascent fecal microbiota from 20 subjects from the Canadian Healthy Infant Longitudinal Development (CHILD) Study using high-throughput sequence analysis of portions of the 16S rRNA gene. Despite significant differences between the dust and fecal microbiota revealed by Nonmetric Multidimensional Scaling (NMDS) analysis, permutation analysis confirmed that 14 bacterial OTUs representing the classes Actinobacteria (3), Bacilli (3), Clostridia (6) and Gammaproteobacteria (2) co-occurred at a significantly higher frequency in matched dust-stool pairs than in randomly permuted pairs, indicating an association between these dust and stool communities. These associations could indicate a role for the indoor environment in shaping the nascent gut microbiota, but future studies will be needed to confirm that our findings do not solely reflect a reverse pathway. Although pet ownership was strongly associated with the presence of certain genera in the dust for dogs (Agrococcus, Carnobacterium, Exiguobacterium, Herbaspirillum, Leifsonia and Neisseria) and cats (Escherichia), no clear patterns were observed in the NMDS-resolved stool community profiles as a function of pet ownership.

Sunbed use and campaign initiatives in the Danish population, 2007-2009: a cross-sectional study.

BACKGROUND: Denmark has experienced an increase in melanoma incidence since the 1960s. In 2007, a skin cancer prevention campaign was launched, one of the targets being the widespread use of sunbeds in Denmark. The antisunbed campaign comprised public affairs initiatives and campaign activities, which included the social media, with young people as the main target. OBJECTIVE: The aim of this study was to describe the development in sunbed use after the start of the campaign in the period 2007-2009. METHODS: A population-based sample of 14,514 respondents aged 15-59 years completed four questionnaires in 2007-2009 on artificial exposure to ultraviolet radiation. We examined the relations between sunbed use, time and demographic factors using logistic regression analysis. RESULTS: The odds ratio (OR) for being a sunbed user in 2009 when compared with 2007 was 0.61 (0.54-0.69); in the age group of 15-19 years, the OR was 0.42 (0.30-0.69). In 2009, however, 23% of Danes (33% of 15-19-year-olds) still reported sunbed use within the past 12 months, and more than 50% had experienced sunburn caused by a sunbed. In 2009, the majority of the population, including the age group of 15-19 years, was in favour of restricting admission to sunbed parlours for children under 18 years. CONCLUSIONS: Sunbed use in Denmark decreased concurrently with the campaign activities, with the largest change in the youngest age group, which was a prioritized target of the campaign. Results suggest that a legislative solution should be found to avoid exposure of a large proportion of children to ultraviolet radiation and to reduce future melanoma incidence.

[Transient splenial lesion in influenza A H1N1 2009 infection]. / Transiente Spleniumläsion bei Influenza-A-H1N1-09-Infektion.

Severe neurologic complications have been rarely reported during novel pandemic influenza A(H1N1) virus infections. We describe the case of an 10-year-old boy with new onset seizures and proven influenza A(H1N1) 2009 infection showing a reversible hyperintense lesion in the splenium of the corpus callosum on T2-weighted and FLAIR magnetic resonance images without contrast enhancement. Transient splenial lesions have been described in the context of virus encephalopathy and do not require specific treatment.

Fish response to the temporal hierarchy of the natural flow regime in the Daly River, northern Australia.

In this study, relationships between flow variation across multiple temporal scales and the distribution and abundance of three fish species, western rainbowfish Melanotaenia australis, sooty grunter Hephaestus fuliginosus and barramundi Lates calcarifer were examined at eight sampling reaches in the Daly River, Northern Territory, Australia. Discharge was highly seasonal during the study period of 2006-2010 with a distinct wet-dry discharge pattern. Significant catchment-wide correlations were identified between species abundance and hydrologic variables across several scales describing the magnitude and variability of flow. A Bayesian hierarchical model which accounted for >80% of variation in abundances for all species and age classes (i.e. juvenile and adult), identified the extent to which the influence of short-term flow variation was dependent upon the historical flow regime. There were distinct ontogenetic differences in these relationships for H. fuliginosus, with variability of recent flows having a negative effect on juveniles which was stronger at locations with higher historical mean daily flow. Lates calcarifer also displayed ontogenetic differences in relationships to flow variation with adults showing a positive association with increase in recent flows and juveniles showing a negative one. The effect of increased magnitude of wet-season flows on M. australis was negative in locations with lower historical mean daily flow but positive in locations with higher historical mean daily flow. The results highlighted how interactions between multiple scales of flow variability influence the abundance of fish species according to their life-history requirements.

Widespread omnivory and low temporal and spatial variation in the diet of fishes in a hydrologically variable northern Australian river.

This article examines the trophic ecology of freshwater fishes (22 species in 15 families) in a wet and dry tropical Australian river of high intra-annual and interannual hydrological variability. Seven major trophic groups were identified by cluster analysis; however, four food items (filamentous algae, chironomid larvae, Trichoptera larvae and Ephemeroptera nymphs) comprised almost half of the average diet of all species. The influence of species, fish size, spatial effects and temporal effects on food use was investigated using redundancy analysis. Size, time and space accounted for little of the perceived variation. Ontogenetic changes in diet were minor and limited to a few large species. Spatial variation in trophic composition of the fish assemblages reflected the effects of the Burdekin Falls and dam, a major geographic barrier, on species distributions. Little spatial variation in diet was detected after accounting for this biogeographical effect. Temporal variations in flow, although marked, had little effect on variations in fish diet composition due to the low temporal diversity of food resources in physically monotonous sand and gravel channels. Species identity accounted for<50% of the observed variation in food choice; omnivory and generalism were pronounced. The aquatic food web of the Burdekin River appears simple, supported largely by autochthonous production (filamentous and benthic microalgae, and to some extent, aquatic macrophytes). Allochthonous food resources appear to be unimportant. The generalist feeding strategies, widespread omnivory and absence of pronounced trophic segregation reported here for Burdekin River fishes may be common to variable and intermittent rivers of subtropical and tropical northern Australia with similar fish communities and may be a general feature of rivers of low habitat diversity and characterized by flow regimes that vary greatly both within and between years.

Evaluation of a large-scale tuberculosis contact investigation in the Netherlands.

The aim of the present study was to evaluate yield and effectiveness of a large-scale contact investigation around a supermarket employee with infectious tuberculosis. Supermarket customers were screened by tuberculin skin test (TST) and/or radiography, depending on individual characteristics. The number of recent infections was estimated based on historical reference data after correction for false-positive TST results. TST screening of 15,518 subjects yielded 12 cases of tuberculosis disease as a direct result of the investigation (1,293 screenings per case identified). Radiographical screening of 5,945 subjects yielded no cases. There were 359 (2.6%) positive TSTs; 117 (34%) were estimated to be due to recent exposure. The number of customers screened in order to find one case of recent infection was 114, varying from 43 for customers who visited the supermarket twice per week or more, to 4,148 for customers who visited less than once per month. In conclusion, although this patient probably transmitted Mycobacterium tuberculosis to at least 117 customers, the contact investigation was inefficient, as large numbers of customers had to be screened and the majority of identified tuberculosis infections were probably not related to the index case. The efficiency could have been improved by omitting radiographical screening and limiting tuberculin skin test screening to customers who reported frequent supermarket visits.

Interferon-gamma release assays during follow-up of tuberculin skin test-positive contacts.

SETTING: Following a large-scale contact investigation, individuals with a positive tuberculin skin test (TST) result were offered preventive tuberculosis treatment. OBJECTIVE: To investigate the effect of isoniazid (INH) treatment and the effect of time on interferon gamma release assay (IGRA) results during follow-up. DESIGN: TST-positive subjects (n = 122) detected during the large-scale contact investigation were included in the study. Blood was obtained every 6 months over 2 years to perform both tests. RESULTS: Preventive INH treatment was completed by 36 of the 122 (29.5%) subjects, 71 (58.2%) were followed up with 6-monthly X-ray screening and 15 (12.3%) did not complete INH treatment. The overall percentage of individuals with a positive result remained stable during the 2 years, at approximately 45-50%, but individual responses varied over time. The majority of initially low IGRA results remained below the cut-off value, initially high IGRA results remained positive, while initially intermediate IGRA results were followed by more dynamic patterns. CONCLUSION: This study showed a highly variable pattern of IGRA responses over time and suggests limited value for their use during follow-up of latently infected individuals. However, the significance of different kinetic patterns observed among subjects with intermediate initial IGRA results warrants further study.

Reliability and consistency of a validated sun exposure questionnaire in a population-based Danish sample.

An important feature of questionnaire validation is reliability. To be able to measure a given concept by questionnaire validly, the reliability needs to be high. The objectives of this study were to examine reliability of attitude and knowledge and behavioral consistency of sunburn in a developed questionnaire for monitoring and evaluating population sun-related behavior. Sun related behavior, attitude and knowledge was measured weekly by a questionnaire in the summer of 2013 among 664 Danes. Reliability was tested in a test-retest design. Consistency of behavioral information was tested similarly in a questionnaire adapted to measure behavior throughout the summer. The response rates for questionnaire 1, 2 and 3 were high and the drop out was not dependent on demographic characteristic. There was at least 73% agreement between sunburns in the measurement week and the entire summer, and a possible sunburn underestimation in questionnaires summarizing the entire summer. The participants underestimated their outdoor exposure in the evaluation covering the entire summer as compared to the measurement week. The reliability of scales measuring attitude and knowledge was high for majority of scales, while consistency in protection behavior was low. To our knowledge, this is the first study to report reliability for a completely validated questionnaire on sun-related behavior in a national random population based sample. Further, we show that attitude and knowledge questions confirmed their validity with good reliability, while consistency of protection behavior in general and in a week's measurement was low.

Neutrophil chemotaxis induced by the diacylglycerol kinase inhibitor R59022.

The diacylglycerol kinase inhibitor R59022 induced chemotaxis in neutrophils. The response to R59022 was primarily chemotactic and only very little chemokinetic. Pretreatment with the protein kinase C inhibitors staurosporine and AMG-C16 inhibited chemotaxis induced by R59022 indicating the involvement of protein kinase C. In contrast, chemotaxis induced by fMet-Leu-Phe was only slightly inhibited by staurosporine and AMG16. The effects of R59022 were comparable to the effects of the protein kinase C activators DiC8 and PMA and suggest an involvement of protein kinase C. Pretreatment with pertussis toxin inhibited R59022-induced migration, fMet-Leu-Phe-induced migration, and random migration. GTP gamma S, which stimulates migration of electropermeabilized neutrophils by itself, causes an additive increase of migration in electropermeabilized neutrophils stimulated with a suboptimal concentration R59022, but causes a synergistic increase of migration in cells stimulated with a suboptimal concentration fMet-Leu-Phe. The effects of GTP gamma S on migration are completely inhibited by AMG-C16. This suggests that the GTP-binding protein involved in R59022-activated migration is the G protein that is associated with random migration.

GTP[S] stimulates migration of electropermeabilized neutrophils via a pertussis toxin-sensitive G-protein.

Electropermeabilized neutrophils were used to study the role of G-proteins in neutrophil migration. Rabbit neutrophils, under specific conditions, retained their ability to migrate after electropermeabilization. Introduction of guanosine-5'-[3-thio] triphosphate (GTP[S]) into the cell interior stimulated random migration and enhanced migration activated by a suboptimal concentration of formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) (10(-11) M). GTP[S] had no effect on random migration by intact cells, or on migration of intact cells activated with a suboptimal concentration of fMet-Leu-Phe, indicating that the effect of GTP[S] was intracellular. The effects of GTP[S] were inhibited by pertussis toxin and by guanosine-5'-[2-thio] diphosphate (GDP beta S) indicating that a pertussis toxin-sensitive G-protein was involved. GTP stimulated random migration to the same extent as GTP[S], but had only a small effect on migration activated by a suboptimal concentration of fMet-Leu-Phe (10(-11) M). Several other nucleotides tested had no effect on random migration or migration activated with 10(-11) M fMet-Leu-Phe. The results show that neutrophil migration can be potentiated by direct activation of a pertussis toxin-sensitive G-protein, and the results obtained with GTP suggest that possibly more than one G-protein is involved in this process.

Modulation of neutrophil migration by exogenous gaseous nitric oxide.

We studied the effect of exogenous nitric oxide (NO) on migration of rabbit peritoneal neutrophils. Exogenous NO enhanced random migration of neutrophils in a concentration-dependent way. An optimally stimulatory effect was observed with 0.5 microM NO, whereas at higher NO concentrations the enhancing effect decreased again. NO caused a rapid and transient increase in intracellular guanosine-3',5'-cyclic monophosphate (cGMP) levels. The enhancing effect of NO on random migration was largely reversed by the inhibitors of cGMP accumulation, LY-83583 and methylene blue, and by the antagonists of cGMP-dependent protein kinase, 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS) and 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS). These observations strongly suggest that the enhancement of random migration by NO is mediated by cGMP and cGMP-dependent protein kinase. The effect of NO on migration did not occur in the absence of extracellular calcium. Although NO did not induce a measurable elevation of intracellular free calcium, pre-incubation with the intracellular calcium chelator Fura-2/AM abolished the enhancing effect of NO. It appears therefore that a small change in the level of cytoplasmic free calcium does play a role in the enhancement of random migration by NO. High concentrations of NO were found to inhibit chemotaxis induced by an optimal concentration of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). This inhibitory effect was also dependent on the presence of extracellular calcium. A role for cGMP in the inhibition of fMLP-induced chemotaxis by NO is not supported by our measurements of intracellular cGMP levels. In contrast to the effects on fMLP, NO did not affect chemotaxis induced by the phorbol ester PMA. In conclusion, we show that NO, not derived from NO donors but applied directly, may stimulate or inhibit neutrophil migration, dependent on the concentration. The enhancing effect of NO on random migration is mediated by cGMP, which emphasizes the importance of this second messenger as a modulator of neutrophil functional.

Preparation of proteosome-based vaccines. Correlation of immunogenicity with physical characteristics.

In order to facilitate the use of proteosome-based vaccines, we have identified and analyzed the parameters that affect their immunogenicity. As a model system we used synthetic peptides (LCF6) containing sequences from the immunodominant (NANP)n tandem repeat region of the P. falciparum circumsporozoite protein, hydrophobically complexed to multimeric protein preparations (proteosomes) of meningococcal outer membrane proteins (OMP), since we have previously shown that high levels of anti-(NANP)n IgG can be elicited in mice by use of this novel adjuvant system (Lowell et al., 1988a). We have now examined these preparations by velocity sedimentation and measured their ability to elicit an IgG response in mice. Velocity sedimentation of freshly mixed OMP and LCF6, without dialysis, produced a limited number of small complexes, whereas dialysis of the mixture for 4 d yielded heterogeneously sized complexes that became more homogeneous when the dialysis was carried out for 7 or 10 days. The most homogeneous of these peptide-proteosome complexes (those dialyzed for 10 days) induced substantial levels of anti-(NANP)n IgG in mice, and shorter periods of dialysis resulted in vaccines that induced proportionately lower titers. Analysis of a series of preparations with varying LCF6: OMP ratios (w/w) showed that the degree of peptide substitution of the proteosomes was inversely proportional to the rate of sedimentation of the complexes and that there exists an optimal degree of lipopeptide complexing to the proteosomes. Our results suggest that the parameters affecting the immunogenicity of the peptide-proteosome complexes are: (i) hapten density, and (ii) size of the complex. Furthermore, sedimentation analysis of peptide-proteosome immunogens may serve as a rapidly performed assay of immunogenic potency.

Identification and localisation of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni.

An abundant 0.9 kb female-specific mRNA in Schistosoma mansoni is thought to code for an egg-shell precursor protein [Bobek et al. (1986) Proc. Natl. Acad. Sci. USA 83, 5544-5548]. This gene contains two ORFs. A recombinant plasmid was constructed that expresses a fusion protein containing a glycine- and tyrosine-rich polypeptide coded for by one of these ORFs. Antisera raised against homogenates of female, but not of male, S. mansoni recognise this fusion protein, providing direct evidence that this ORF is used by S. mansoni. In comparative Western blots of S. mansoni homogenates from males and females affinity purified antibodies that react with the fusion protein react exclusively with proteins from females, recognising a 28 kDa polypeptide and a smear of immunoreactive material probably caused by oxidative crosslinking. In immunohistology, the affinity purified antibodies react with mature vitelline cells in female schistosomes. The immunoreactive material is localised in the so-called 'vitelline droplets' that are morphologically very similar to 'shell globules', known to contain egg-shell precursors, that are found in Fasciola hepatica. In situ hybridisation shows that the eggshell precursor gene is only transcribed in immature vitelline cells and has a short half-life. Taken together, these observations provide persuasive evidence that the 0.9 kb mRNA codes for an eggshell precursor.

The stimulation of human neutrophil migration by angiotensin IL: its dependence on Ca2+ and the involvement of cyclic GMP.

1. Angiotensin II had a bimodal effect on human neutrophil migration. Low concentrations of angiotensin II stimulated random migration. At a concentration of 10(-10) M it caused a maximal increase of migration; migration increased from 47.2 +/- 2.1 microns in the absence of angiotensin II, to 73.1 +/- 2.2 microns with 10(-10) M angiotensin II present in the lower compartment of the Boyden chamber (n = 5, P < 0.001). Stimulation of migration by angiotensin II was partly chemotactic and partly chemokinetic. Angiotensin II concentrations of 10(-8) M and higher inhibited chemotactic peptide-stimulated chemotaxis. 2. The stimulant effect of angiotensin II on migration was completely dependent on extracellular Ca2+. In the presence of 1 mM Ca2+, angiotensin II stimulated migration to 76.1 +/- 1.7 microns, while migration in the absence of Ca2+ was 42.2 +/- 1.9 microns (n = 4, P < 0.001). Different types of calcium channel blockers either moderately or strongly inhibited angiotensin II-activated migration. Stimulation of migration by angiotensin II in intact cells required higher concentrations of Ca2+ than in electroporated cells. This supports the view that there is an influx of Ca2+ through the plasma membrane, and a requirement of calcium for an intracellular target. 3. Angiotensin II-stimulated migration was inhibited by pertussis toxin; from 71.6 +/- 2.0 microns in the absence, to 43.6 +/- 1.5 microns in the presence of pertussis toxin (n = 4, P < 0.001). Migration of electroporated neutrophils stimulated by angiotensin II was synergistically enhanced by GTP gamma S. This suggests that one or more G-proteins are involved in the activating effect of angiotensin II. 4. Inhibitors of soluble guanylate cyclase and antagonists of cyclic GMP-dependent kinase strongly inhibited the activating effect of angiotensin II. The results suggest that the activating effect of angiotensin II is mediated by cyclic GMP and by cyclic GMP-dependent kinase.

Endothelin-induced activation of neutrophil migration.

Migration of rabbit peritoneal neutrophils was stimulated by endothelin-1 (ET-1) up to 2.10(-8) M. Higher concentrations inhibited random migration. The stimulating effect of ET-1 was inhibited by BQ-123, a specific antagonist of the ETA receptor. A checkerboard assay showed that the stimulating effect of ET-1 on neutrophil migration was chemokinetic rather than chemotactic. Extracellular Ca2+ was required for the activating effect of ET-1. Non-selective calcium channel blockers such as econazole and La3+ strongly inhibited ET-1-activated migration but had little effect on fMLP-activated migration, underlining the importance of Ca2+ influx for ET-1-activated migration. Studies with electroporated neutrophils showed that the increase in migration was most pronounced at calcium concentrations between 100 nM and 1 microM. ET-1-activated migration of electroporated cells was completely blocked by low concentrations of calcium-channel blockers such as verapamil and nitrendipine. Migration by intact cells was inhibited by the same concentration of verapamil, but to a lesser degree; nitrendipine had little effect on migration of intact cells. This suggests that calcium derived from intracellular stores is required for migration activated by ET-1. Protein kinase C, protein tyrosine kinase, and phosphatase activity were involved in the activating effect of ET-1 on neutrophil migration. ET-1 did not induce exocytotic enzyme release, in neither the presence nor the absence of cytochalasin B.

Ryanodine as inhibitor of chemotactic peptide-induced chemotaxis in human neutrophils.

Ryanodine gave a moderate inhibition of chemotactic peptide-activated chemotaxis by intact human neutrophils. Chemotaxis by electroporated neutrophils was strongly inhibited in the nanomolar concentration range. Inhibition of chemotaxis by electroporated neutrophils occurs at concentrations known to open calcium channels in ryanodine-sensitive Ca2+ stores. Whereas migration by formyl-methionyl-leucyl-phenylalanine (fMLP)- or interleukin-8-activated electroporated neutrophils was strongly inhibited by ryanodine, chemotaxis induced by protein kinase C activators was not affected. This suggests that the importance of ryanodine-sensitive Ca2+ stores for migration depends on the type of activator used. Ryanodine gave an increase of cytoplasmic free calcium due to the liberation of calcium from internal stores and to the influx of extracellular calcium. The results show that the neutrophil contains ryanodine-sensitive calcium stores that might be involved in receptor-mediated chemotaxis.

Inhibition of interleukin-8-activated human neutrophil chemotaxis by thapsigargin in a calcium- and cyclic AMP-dependent way.

Chemotactic migration of human neutrophils, induced by interleukin-8 (IL-8) or other activators, was inhibited by thapsigargin in the high nanomolar range. The degree of inhibition depended on the type of activator. Other inhibitors of Ca(2+)-ATPases associated with intracellular calcium stores, such as cyclopiazonic acid and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, equally inhibited IL-8-activated migration. Inhibition of migration by thapsigargin and the other ATPase inhibitors occurred only in the presence of extracellular Ca2+; migration was not inhibited in the presence of EGTA. La3+ reversed thapsigargin-induced inhibition to a large degree; other calcium channel blockers gave a partial reversal (econazole, verapamil, and SK&F 96365) or had no effect (gadolinium chloride and Ni2+). Using electroporated cells and Ca buffers, it was shown that inhibition started at about 0.2 microM and was complete at a cytosolic Ca concentration of about 2 microM. It appears that under certain conditions the thapsigargin-induced influx of extracellular calcium, causing relatively high local calcium concentrations, initiates or permits a process which may be detrimental to chemotactic migration. Cyclic AMP (cAMP; adenosine 3',5'-cyclic monophosphate) is probably involved in this process, because thapsigargin increased the cAMP level and cAMP inhibited IL-8-activated migration in a calcium-dependent way. The hypothesis that cAMP is involved in the effect of thapsigargin on migration is supported by the finding that very low concentrations of thapsigargin stimulate neutrophil migration in the absence of other chemoattractants. The results suggest that thapsigargin causes a (compartmentalized) increase in cAMP, which results in a calcium-dependent modulation of migration.