Evaluation of serum IgE in peach-allergic patients with systemic reaction by using recombinant Pru p 7 (gibberellin-regulated protein).

BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n=27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n=17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P<0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction.

Abnormal asymmetries in subcortical brain volume in schizophrenia.

Subcortical structures, which include the basal ganglia and parts of the limbic system, have key roles in learning, motor control and emotion, but also contribute to higher-order executive functions. Prior studies have reported volumetric alterations in subcortical regions in schizophrenia. Reported results have sometimes been heterogeneous, and few large-scale investigations have been conducted. Moreover, few large-scale studies have assessed asymmetries of subcortical volumes in schizophrenia. Here, as a work completely independent of a study performed by the ENIGMA consortium, we conducted a large-scale multisite study of subcortical volumetric differences between patients with schizophrenia and controls. We also explored the laterality of subcortical regions to identify characteristic similarities and differences between them. T1-weighted images from 1680 healthy individuals and 884 patients with schizophrenia, obtained with 15 imaging protocols at 11 sites, were processed with FreeSurfer. Group differences were calculated for each protocol and meta-analyzed. Compared with controls, patients with schizophrenia demonstrated smaller bilateral hippocampus, amygdala, thalamus and accumbens volumes as well as intracranial volume, but larger bilateral caudate, putamen, pallidum and lateral ventricle volumes. We replicated the rank order of effect sizes for subcortical volumetric changes in schizophrenia reported by the ENIGMA consortium. Further, we revealed leftward asymmetry for thalamus, lateral ventricle, caudate and putamen volumes, and rightward asymmetry for amygdala and hippocampal volumes in both controls and patients with schizophrenia. Also, we demonstrated a schizophrenia-specific leftward asymmetry for pallidum volume. These findings suggest the possibility of aberrant laterality in neural pathways and connectivity patterns related to the pallidum in schizophrenia.

High-resolution record of Northern Hemisphere climate extending into the last interglacial period.

Two deep ice cores from central Greenland, drilled in the 1990s, have played a key role in climate reconstructions of the Northern Hemisphere, but the oldest sections of the cores were disturbed in chronology owing to ice folding near the bedrock. Here we present an undisturbed climate record from a North Greenland ice core, which extends back to 123,000 years before the present, within the last interglacial period. The oxygen isotopes in the ice imply that climate was stable during the last interglacial period, with temperatures 5 degrees C warmer than today. We find unexpectedly large temperature differences between our new record from northern Greenland and the undisturbed sections of the cores from central Greenland, suggesting that the extent of ice in the Northern Hemisphere modulated the latitudinal temperature gradients in Greenland. This record shows a slow decline in temperatures that marked the initiation of the last glacial period. Our record reveals a hitherto unrecognized warm period initiated by an abrupt climate warming about 115,000 years ago, before glacial conditions were fully developed. This event does not appear to have an immediate Antarctic counterpart, suggesting that the climate see-saw between the hemispheres (which dominated the last glacial period) was not operating at this time.

Characteristics of Diffusional Kurtosis in Chronic Ischemia of Adult Moyamoya Disease: Comparing Diffusional Kurtosis and Diffusion Tensor Imaging.

BACKGROUND AND PURPOSE: Detecting microstructural changes due to chronic ischemia potentially enables early identification of patients at risk of cognitive impairment. In this study, diffusional kurtosis imaging and diffusion tensor imaging were used to investigate whether the former provides additional information regarding microstructural changes in the gray and white matter of adult patients with Moyamoya disease. MATERIALS AND METHODS: MR imaging (diffusional kurtosis imaging and DTI) was performed in 23 adult patients with Moyamoya disease and 23 age-matched controls. Three parameters were extracted from diffusional kurtosis imaging (mean kurtosis, axial kurtosis, and radial kurtosis), and 4, from DTI (fractional anisotropy, radial diffusivity, mean diffusivity, and axial diffusivity). Voxelwise analysis for these parameters was performed in the normal-appearing brain parenchyma. The association of these parameters with neuropsychological performance was also evaluated. RESULTS: Voxelwise analysis revealed the greatest differences in fractional anisotropy, followed, in order, by radial diffusivity, mean diffusivity, and mean kurtosis. In patients, diffusional kurtosis imaging parameters were decreased in the dorsal deep white matter such as the corona radiata and superior longitudinal fasciculus (P < .01), including areas without DTI abnormality. Superior longitudinal fasciculus fiber-crossing areas showed weak correlations between diffusional kurtosis imaging and DTI parameters compared with tissues with a single-fiber direction (eg, the corpus callosum). Diffusional kurtosis imaging parameters were associated with general intelligence and frontal lobe performance. CONCLUSIONS: Although DTI revealed extensive white matter changes, diffusional kurtosis imaging additionally demonstrated microstructural changes in ischemia-prone deep white matter with abundant fiber crossings. Thus, diffusional kurtosis imaging may be a useful adjunct for detecting subtle chronic ischemic injuries.

Nucleotide sequence of rat erythropoietin.

The cDNA for the rat erythropoietin (EPO) has been cloned and sequenced. The deduced amino acid sequence consists of 166 amino acid residues, which has a 79% and 95% homology with human and mouse EPOs, respectively. Many short stretches, highly conserved in primate and rodent EPOs, are found in the 3'-noncoding region when insertions and deletions are taken into consideration.

Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102.

The crystal structure of an enzyme having polychlorinated-biphenyl degrading activity, the BphC enzyme from Pseudomonas sp. strain KKS102, has been solved as a free form at 1.8 A resolution. This is the first three-dimensional structure among the extradiol-type dioxygenases. Based on 34,387 reflections (10.0 to 1.8 A, completeness 87.8%), a current R-factor of 20.4% (with a free R-factor of 24.3%) was obtained with a model obeying standard geometry within 0.011 A in bond lengths and 1.91 degrees in bond angles. The BphC enzyme is a homo-octamer and each subunit is composed of two domains: Domain 1 (N-terminal part) and Domain 2 (C-terminal part). Each domain contains two repetitions of a novel folding motif (the "beta alpha beta beta beta" motif) each consisting of ca 55 amino acid residues. A single Fe ion in the active site coordinates the side-chains of three amino acid residues (His145, His209 and Glu260) and two solvent molecules. The coordination geometry is that of a square pyramid. In addition to the free form of the BphC enzyme, we have solved two three-dimensional structures of the BphC enzyme complexed with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3-methylcatechol (3-MCT). These substrates were found intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present crystals. In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side-chain ligands, were found to form a penta-coordinated system around the Fe ion roughly arranged in a trigonal bipyramidal configuration. The active site structures appear to be essentially consistent with the reaction mechanism proposed so far.

Role of hydrophilic organic matter on developing toxicity in decay process of activated sludge.

It is known that the toxicity of effluent is more intensive than that of influent in the activated sludge process. In this study, we applied bioassay using cultured human cell lines to the decay process of activated sludge to evaluate the toxicity of organic matter generated and/or released from activated sludge bacteria. We also applied this bioassay to hydrophilic fraction of samples. The bioassay results showed that: (1) the variation in the dose-response relation obtained from assay with original samples was observed during decay; (2) on the other hand, the response curves of only hydrophilic fraction at each time show the same relationship between TOC and viability of MCF7 cells; (3) this trend was confirmed by plotting the time course of EC50. These results imply that: (1) the hydrophilic organic matter controlled for developing toxicity during decay process of activated sludge; and (2) the character of hydrophilic organic matter is not changed during the experimental period.

Organic matter released from activated sludge bacteria cells during their decay process.

Organic matter released from activated sludge bacteria is a considerable issue in the wastewater reclamation process. In this study, we focused 2-keto-3-deoxyoctulosonic acid in the Lipopolysaccharide existed in the gram-negative bacterial cell wall as an index of organic matter released from bacteria, and investigated the fate of 2-keto-3-deoxyoctulosonic acid in the aerated and ultrasonicated activated sludge samples. The results shows 1) 2-keto-3-deoxyoctulosonic acid concentration in the hydrolyzed sample was higher than non-hydrolyzed sample, and this implied that 2-keto-3-deoxyoctulosonic acid existed in the water phase as a monomer and also as a polymer such as Lipopolysaccharide form and their fragments; 2) the value of (2-keto-3-deoxyoctulosonic acid)/(dissolved organic carbon) ratio did not change in the sludge sonication process and was approximately 0.0006, on the other hand, in the bacteria decay process, the ratio varied from zero to approximately 0.0012; 3) the linear relationship was observed between the degraded heterotrophic biomass and the generated 2-keto-3-deoxyoctulosonic acid; and 4) 2-keto-3-deoxyoctulosonic acid might be considered as an index of organic matter originated from activated sludge bacteria cell.

Retinoic acid up-regulates erythropoietin production in hepatoma cells and in vitamin A-depleted rats.

Retinoic acid (RA) stimulated the production of erythropoietin (Epo) in a human hepatoma cell line, HepG2 cells. The stimulation was due to the accumulation of Epo mRNA. The Epo production in HepG2 cells was also dependent on O2 tension for cell culture but the enhancement of Epo production by RA was independent of O2 tension, indicating that RA exerts its effect through a pathway different from O2. Oral administration of RA to the vitamin A-depleted rats elevated the concentration of Epo in serum. These results suggest that RA up-regulates EPO production in vivo as well as in vitro.

Clinical significance of reverse redistribution on 24-hour delayed imaging of exercise thallium-201 myocardial SPECT: comparison with myocardial fluorine-18-FDG-PET imaging and left ventricular wall motion.

UNLABELLED: Clinical significance of reverse redistribution on 24-hr delayed images after exercise 201Tl myocardial SPECT was investigated in 16 patients with recent myocardial infarction. METHODS: Findings of 24-hr delayed 201Tl SPECT imaging were compared with those of glucose-loaded 18F-fluorodeoxyglucose (FDG) imaging by myocardial PET and with left ventricular wall motion obtained by bi-plane contrast left ventriculography. In each patient, transaxial thallium images and corresponding 18F-FDG images were divided into five ROIs. RESULTS: Reverse redistribution was found in 15 of 80 regions. The mean FDG activity score in regions with reverse redistribution was significantly lower than that in regions having normal or slightly decreased thallium activity on 24-hr delayed imaging; it was significantly higher than that in regions having severely decreased or no thallium activity on 24-hr delayed imaging. The mean wall motion score in regions with reverse redistribution was significantly lower than in regions with normal or slightly decreased thallium activity, however, it was significantly higher than that in regions with moderately or more decreased thallium activity. CONCLUSION: These findings demonstrate that in regions showing reverse redistribution on 24-hr delayed 201Tl imaging, myocardial exogenous glucose utilization and left ventricular wall motion had deteriorated, but were not on a level with the scar.

Calcium entry blockers: antihypertensive and natriuretic effects in experimental animals.

Hypotensive and natriuretic effects of calcium entry blockers were studied in spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKYs). In SHRs, the hypotensive action of diltiazem was enhanced, while that of hydralazine was not different from its action in WKYs. Diltiazem, unlike hydralazine and nifedipine, did not cause reflex tachycardia in rats. Diltiazem and nifedipine caused an increase in urine volume and sodium excretion. The natriuretic potency of diltiazem was the same in both SHRs and WKYs and was not affected by pretreatment with indomethacin. Diltiazem increased plasma renin activity but had no influence on plasma aldosterone concentration. Hydralazine increased both plasma renin activity and plasma aldosterone concentration and decreased sodium excretion. In anesthetized dogs, diltiazem increased sodium excretion, glomerular filtration rate and renal blood flow. Diltiazem may have a direct effect on tubular sodium reabsorption. This assumption was supported by a study with short-circuit current of the isolated bullfrog bladder. It is likely that diltiazem's effect of increasing urine volume and sodium excretion is due mainly to changes in renal hemodynamics and partly to direct action on renal tubules. Diltiazem opposed angiotensin II-induced responses, such as renal vasoconstriction and reduction of glomerular filtration rate. Under this condition, a marked natriuretic effect was observed with both intravenous and intraarterial administration of diltiazem. The ability to increase urinary excretion of sodium is a desirable characteristic for an antihypertensive agent. Calcium entry blockers have this ability and are therefore able to act without causing sodium and water retention.

Evaluation of left ventricular early diastolic performance by color tissue Doppler imaging of the mitral annulus.

A noninvasive assessment of left ventricular (LV) diastolic performance by tissue Doppler imaging was performed in 56 patients (8 patients with atypical chest pain, 42 with coronary artery disease with a previous myocardial infarction, and 6 without a previous myocardial infarction) who underwent cardiac catheterization. Mitral annular velocity (MAV) during early ventricular diastole was obtained by M-mode color tissue Doppler imaging at the posterior corner of the mitral annulus. In each patient, the negative peak of the first derivative of LV pressure decay (peak -dP/dt) and a time constant of LV relaxation (tau) were calculated from the LV pressure waves obtained by a catheter-tip micromanometer. LV end-systolic volume index was measured from contrast left ventriculography. MAV during early diastole was significantly correlated with tau (r = -0.73, p <0.001), peak -dP/dt (r = 0.58, p <0.001), and LV end-systolic volume index (r = -0.63, p <0.001). On multivariate regression analysis with MAV during early diastole, tau and LV end-systolic volume index were selected as prime determinants (r = 0.80, p <0.001). These findings suggest that MAV during early diastole has a direct relation to LV elastic recoil as well as to LV relaxation. MAV during early diastole gives important information regarding LV behavior in late systole to early diastole where LV early diastolic performance is determined.

Selective and full beta 1-adrenoceptor agonist action of a catechol derivative of denopamine (T-0509) in the guinea-pig cardiac muscle and trachea: comparison with denopamine, xamoterol and isoprenaline.

1. The pharmacological actions of T-0509, a 3-hydroxy derivative of denopamine, were studied in various guinea-pig tissues; these effects were compared with those of isoprenaline, denopamine and xamoterol. 2. The intrinsic activities of the positive inotropic actions of T-0509, denopamine and xamoterol compared with isoprenaline (= 100%) in the papillary muscle were 99%, 83% and 28%, respectively, while their relative potencies (EC50 agonist EC50 isoprenaline) were 0.23, 33 and 1.4, respectively. The intrinsic activities of T-0509, denopamine and xamoterol as positive chronotropic agents in the right atria were 98%, 69% and 48%, respectively, and their equipotent concentrations (isoprenaline = 1) were 0.24, 50 and 4, respectively. 3. The positive chronotropic actions of T-0509 and denopamine were antagonized by bisoprolol (3 x 10(-8) M), but not by ICI 118,551 (3 x 10(-8) M). 4. The intrinsic activity of T-0509 in histamine-contracted tracheae was similar to that of isoprenaline, but its equipotent concentration was 38; the effects of both agents were antagonized by ICI 118,551 (3 x 10(-8) M), but not by bisoprolol (3 x 10(-8) M). Denopamine and xamoterol did not show any agonist activity on guinea-pig trachea. 5. Denopamine and xamoterol antagonized the positive chronotropic (pA2, denopamine: 6.98, xamoterol: 7.75) and tracheal relaxant (pA2, denopamine: 5.39, xamoterol: 6.25) effects of isoprenaline. 6. Isoprenaline, T-0509 and denopamine, but not xamoterol, contracted the guinea-pig aorta in a decreasing order in the presence of propranolol (10(-6) M).7. Based on the above studies, T-0509 appears to be a highly selective betaI-adrenoceptor agonist with full agonist properties, while denopamine and xamoterol appear to be selective, but partial betaI-adrenoceptor agonists.

Effects of R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-N,N-diethylamino)ethoxy]ethan ol hydrochloride (T-588), a novel cognitive enhancer, on noradrenaline release in rat cerebral cortical slices.

We investigated the effects of R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino)ethoxy]ethan ol hydrochloride (T-588), a novel cognitive enhancer, on noradrenaline (NA) release from rat cerebral cortical slices in vitro. Addition of T-588 in an assay mixture stimulated [3H]NA release from prelabeled slices in the presence or absence of extracellular CaCl2, and in the presence of the Ca2+/calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. T-588 stimulated NA release with a time lag of about 1 min, and the high level of release was maintained for at least 10 min, whereas maximal KCl-evoked NA release was observed within 1 min after the addition of KCl, and the effect declined subsequently. The effect of T-588 was reversible (pretreatment with T-588 showed no effect on NA release after two washes by centrifugation). We also compared the effects of T-588 and N-ethylmaleimide (NEM), a sulfhydryl alkylating agent known to stimulate neurotransmitter release in several types of cells. The addition of NEM stimulated NA release irreversibly from the slices in a Ca2+-independent manner, and the effect of NEM, but not that of T-588, was inhibited by the simultaneous addition of dithiothreitol, a sulfhydryl group reducing agent. The addition of T-588, which stimulated NA release by itself, inhibited the NA release by 0.6 mM NEM, although the effect of T-588 was additive in the presence of 0.2 mM NEM. These findings suggest that T-588 stimulates NA release from rat cerebral cortical slices in a Ca2+- and calmodulin-independent manner, possibly via an NEM-sensitive factor(s), although the mechanism of the effects of T-588 seems to be different from that of NEM.

Ecabet sodium, a novel locally-acting anti-ulcer agent, protects the integrity of the gastric mucosal gel layer from pepsin-induced disruption in the rat.

BACKGROUND: Ecabet sodium, a novel non-systemic anti-ulcer agent, possesses high affinity to gastric adherent mucus, which plays an important role in the protection of the gastric epithelium against acid and pepsin. AIM: To assess the effect of ecabet on pepsin-induced degradation of the structure of the mucus gel layer. METHODS: Everted sacs of rat stomach were incubated in HCl solution containing pepsin with or without ecabet. Pepsin-induced release of the cleaved peptides and hexosamine from the sacs was determined. Changes in the molecular size of glycoproteins in the adherent mucus (using gel filtration methods) and in the morphology of the epithelium (using both light and scanning electron microscopy) were also examined. RESULTS: Ecabet reduced the pepsin-induced release of peptides and hexosamine, depending on its content in the adherent mucus. Pepsin treatment partially lowered the molecular weight of native glycoproteins in the adherent mucus, caused exfoliation of the epithelial cells, and degraded the network-like ultrastructure of the mucus layer, giving it a lumpy, globular appearance. Ecabet prevented both the pepsin-induced molecular size shift in mucus glycoproteins, and morphological alteration of the epithelium, including ultrastructural derangement of the mucus gel layer. CONCLUSION: Ecabet protects the polymeric structure of mucus glycoproteins from proteolytic degradation by pepsin, and thus maintains integrity of the gastric mucus gel layer.

Production and application of monoclonal antibodies specific to pyrroloquinoline quinone.

We produced five monoclonal antibodies (mAbs 1, 2, 6, 7, and 9) that are specific to pyrroloquinoline quinone (PQQ). PQQ-conjugated hemocyanin was used for the immunization of mice and the hybridomas were selected using PQQ-conjugated BSA in an enzyme-linked immunosorbent assay. MAbs 2 and 9 were of the IgG1 isotype. Both could recognize free PQQ, the former probably at the o-quinone and the latter at the opposite side of the molecule. They did not bind with trihydroxyphenylalanine, dihydroxyphenylalanine, 1,2,4-trihydroxybenzene, ascorbic acid, riboflavin, or menadione. In contrast to the IgGs, mAbs 1, 6, and 7 (IgMs) did not bind with free PQQ. Using mAb 2, a competitive enzyme-linked immunosorbent assay was developed, which enabled us to determine 50 nM-1 microM free PQQ. Furthermore, we analyzed the covalently bound prosthetic groups of two quinoproteins (amine oxidase from Aspergillus niger and amine dehydrogenase from Pseudomonas putida) by Western analysis using these mAbs. However, the results was negative, indicating that the prosthetic groups are not PQQ.

Phospholipid reverse micelles as a milieu of an enzyme reaction in an apolar system.

Alkaline phosphatase was entrapped in reverse micelles formed in n-heptane with surfactants (bis(2-ethylhexyl)sodium sulfosuccinate, phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid) and water. The entrapped enzyme could express its activity in the reverse micelles of the above surfactants only when the substrate, p-nitrophenylphosphate, was within the reverse micelles of bis(2-ethylhexyl)sodium sulfosuccinate. The optimum pH of activity in the reverse micelles was higher by about one pH unit than that determined in bulk water. Regardless of water content (mol water/mol surfactant = 10 or 14.8), the Km value was about 30 mM, while Vmax at the higher water content (14.8) was 2-4 times greater than that at 10. Activation energy of the reaction depended on the kind of reverse micelles. Differences in carbon chain length of solvents showed no effect on the kinetic properties of the enzyme.

A sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography.

A simple and sensitive method was developed for quantitative analysis of phospholipid molecular species. Diacylglycerols were prepared from phospholipids by phospholipase C treatment and converted to the corresponding dinitrobenzoyl derivatives, which could be sensitively detected at 254 nm. The derivatives of 21 molecular species were resolved by high-performance liquid chromatography with an octadecylsilyl reversed-phase column. All the derivatives had the same peak area per mol, and peak areas were proportional to the amounts of the derivatives. Quantification was carried out at the picomole level.

Phosphoinositide breakdown as an indirect link between stimulation and aggregation of rat platelets by thrombin and collagen.

When washed rat platelets (1.5 x 10(9)/ml) were stimulated by a threshold concentration of thrombin (0.3 unit/ml) or collagen (10 micrograms/ml), a lag period of about 10 or 30 s, respectively, was seen before the start of aggregation. During the lag period, [32P]phosphatidylinositol 4,5-bisphosphate was degraded as the earliest event within 5-10 s of addition of the stimulus. However, though the extent of phosphatidylinositol 4,5-bisphosphate degradation within 10 s of addition of collagen was greater than that within 20 s of addition of thrombin (0.3 unit/ml), a lag of about 20 s remained before the initiation of aggregation by collagen. This casts doubt on the hypothesis that the stimulus-dependent phosphatidylinositol 4,5-bisphosphate breakdown induces the aggregation of platelets. Phosphatidylinositol labeled with 32Pi or [1-14C]arachidonic acid was scarcely degraded during the lag period. As aggregation proceeded, [14C-arachidonic acid]phosphatidylinositol was degraded with generation of diacylglycerol, phosphatidic acid, arachidonic acid and its metabolites. The maximum aggregation by collagen of rat platelets in which arachidonic acid of phospholipids was replaced in vivo with eicosapentaenoic acid was reduced, but that by thrombin was not, though reduction of thromboxane A2 generation was caused by both stimuli. Indomethacin also fully inhibited the aggregation induced by collagen, but not that induced by thrombin. Hence, thromboxane A2 is required for full aggregation by collagen, but not that by thrombin. These results indicate that thrombin-induced phosphoinositide metabolism may proceed independently of aggregation.