, 2007 and Pele et al., 2007) reducing their usefulness. Effective food allergen management plans seek to minimise the use of such labels, such as the Voluntary Incidental Trace Allergen Labelling (VITAL) system in place in Australia, (Zurzolo, Mathai, Koplin,

& Allen, NVP-AUY922 chemical structure 2012). Such plans require access to well validated methods of allergen analysis. Currently these are largely lacking, partly because comparison of different allergen detection methodologies, such as enzyme-linked immunosorbent assay (ELISA), polymerase-chain reaction (PCR) based methods, and mass spectrometry based methods, cannot be developed as incurred reference materials are not readily available (Heick et al., 2011, Kerbach et al., 2009 and Taylor et al., 2009). The complexity of food matrices and their lack of stability over long periods of time (ideally several years) when stored at ambient temperature makes the preparation of incurred reference materials problematic.

This is further complicated for allergens where the hazard (and hence the analytical target of choice) is a group of allergenic protein molecules, which are prone to modification, aggregation and interactions with other food components selleckchem (e.g., lipids and starches) in complex processed food matrices (Mills, Sancho, Rigby, Jenkins, & Mackie, 2009). Although certified reference materials, such as egg and skimmed milk powder, are available and are being used as calibrants and standards in commercial assays for allergen analysis, none have been designed specifically for food allergen analysis. Calibrants and reference materials may also contain levels of protein modification atypical for food ingredients such as skimmed milk powder, possibly as a result of γ-irradiation used to extend BCKDHA shelf-life (Johnson, Philo, Watson, & Mills, 2012), and have not been evaluated

with regards their allergenic activity. The ultimate verification of a food ingredient’s allergenic activity is demonstrated by their ability to trigger an allergic reaction in a food allergic individual. A range of food ingredients have been used as the active components in oral food challenge procedures used for diagnosis of food allergies in double blind placebo controlled food challenge (DBPCFC) (Vlieg-Boerstra et al., 2011). DBPCFC was a cornerstone of allergy diagnosis in the EuroPrevall project (FOOD-CT-2005-514000) and made use of a standardised food challenge dessert matrix in which dry powdered food ingredients, including peanut, hazelnut, celery spice, skimmed milk and pasteurised egg white powder, could be blinded (Cochrane et al., 2011, Mackie et al., 2012 and Mills et al., 2007). Comprising cold swelling starch, cocoa powder, sugar and a small amount of corn oil and emulsifier as a texturising agent, powdered allergenic ingredients could be homogeneously incurred into the dessert matrix base, which was then hydrated prior to consumption.

These results show that the tested concentrations of EGCG were not genotoxic, meaning that they did not induce any significant DNA damage in the tested cells. Biotransformation of EGCG with tannase did not alter these results. In summary, our data show that unmodified and biotransformed green tea extracts and EGCG were neither cytotoxic nor genotoxic. Furthermore, we observed that the antioxidant and anti-proliferative capacities of these compounds were significantly increased by enzymatic intervention. Due to the potential cancer INCB024360 chemical structure chemopreventive mechanisms of green tea and EGCG include prevention of DNA damage (Malhomme de la Roche et al., 2010 and Morley et al., 2005),

inhibition of inflammatory processes, decreased angiogenesis, and antiproliferative/pro apoptotic effects (Shimizu et al., 2011 and Yang and Wang, 2011), we used the Human Cancer Pathway

Finder Array to evaluate the effects of unmodified and biotransformed green tea extract and EGCG on the expression profiles of 84 genes representative of the six biological pathways involved in transformation and tumorigenesis. Treatment with either unmodified or biotransformed green tea extract significantly changed the expression of 14% of the tested genes (12/84), whereas treatment with either unmodified or biotransformed EGCG altered the pattern of expression of 17% (14/84) of the genes. The statistically significant and biologically relevant results are shown in Table 4. The gene expression values presented were obtained by normalising expression levels to those selleck kinase inhibitor observed in the control cells. In relation to apoptosis and cell cycle control, our data showed that APAF1 (apoptotic peptidase activating

factor 1), CASP8 (caspase 8, apoptosis-related cysteine peptidase), CDKN1A (cyclin-dependent kinase inhibitor 1A), and FAS (TNF receptor superfamily member 6) were up regulated by biotransformed green tea extract, unmodified Baricitinib EGCG and biotransformed EGCG. We also observed a down regulation of CDK2 and 4 (Cyclin-dependent kinase 2 and 4), bcl2 (B-cell CLL/lymphoma 2), bcl2L1 (BCL2-like-1), E2F1 (E2F transcription factor 1), and c-myc (V-myc myelocytomatosis viral oncogene homologue) (Table 4). APAF1, CASP8 and CDKN1 are closely related to the caspase enzyme family. Some of these genes encode members of the caspase family of proteases, whereas others encode proteins responsible for caspase activation. In either case, these proteins contribute to the initiation of the caspase cascade that commits the cell to apoptosis (Gramantieri et al., 2005, Jones et al., 2011 and Yang et al., 2006). The protein encoded by the FAS gene is a member of the TNF-receptor superfamily. This superfamily includes FAS, CD40, CD27, and RANK. FAS contains a death domain, and the interaction of this receptor with its ligand allows the formation of a death-inducing signalling complex that includes Fas-associated death domain protein (FADD), caspase 8, and caspase 10.

In the US, the Environmental Protection Agency has an endocrine disruptor screening programme to validate methods for estrogenic, androgenic and thyroid hormone-like substances. Despite these initiatives, many open questions remain such as, i) defining ‘endocrine disrupter’ (ED), A closer look at each point of the above points followed. Navitoclax manufacturer i) The International Programme on Chemical Safety and Weybridge have proposed slightly

different definitions of an endocrine disrupter. It has been suggested by ECETOC to adopt the Weybridge definition and this appears likely. Current experience in the EFSA Pesticide Risk Assessment Peer Review (PRAPeR) show several current and pressing needs including a clear definition of endocrine disruption and guidelines to reduce uncertainties, scientific and practical tools, and harmonisation in interim measures. Research is needed to understand the basic science and mechanisms of action, and to develop TSA HDAC price measurement methods and risk assessment models. Screening and testing methodologies have to be developed to identify potential endocrine disrupters and to determine adverse effects and dose–response curves and to assess risk, taking into account the requirements of the current legislation. The presentation concluded with a review of the

next steps to be taken in order to reach a consensus on how to regulate endocrine-active pesticides. First the development and validation of appropriate screens and tests, to be followed by OSBPL9 development of procedures and policies and

finally development of standard evaluations and risk assessment guidelines. This is a big order, but progress has begun. At OECD, new guidelines were adopted in September 2009 which accepted two assays and validated a third. The Hershberger assay (Test Guideline 441) and the Human Estrogen Receptor Transcription Assay (Test Guideline 455) were adopted as standard tests and the Repeat Dose 28-day Oral Toxicity (Test Guideline 407) was updated and validated, although here some further fine tuning may be necessary. Finally an examination of validated Quantitative Structure-Activity Relationships could allow an automated look at numerous compounds without the use of animal studies. Decision Criteria in Human Health Risk Assessment for ED Substances. Dr. Karen Hirsch-Ernst, BfR, Germany. This talk was a summary of the meeting Establishment of assessment and decision criteria in human health risk assessment for substances with endocrine disrupting properties under the EU plant protection product regulation, hosted by the German Federal Institute for Risk Assessment (BfR) in Berlin from 11 to 13 November 2009. This was a preliminary report, reflecting the discussion and part of the results of the BfR workshop, but not necessarily detailing the opinion of all participants or of the institutions they work for.

, 2013). To answer the question of whether the germination rate is influenced negatively SCH772984 supplier by flooding, the germination rate of samaras after storage in water was tested. For this experiment, samaras of different F. pennsylvanica trees from a stand situated in a floodplain forest along the River Elbe near Dessau in Sachsen-Anhalt were again used. The samaras were collected in autumn 2006 and stored dry at 5–8 °C until spring 2008. Only full seeds were used in the test. The average dimensions of the samaras (mean ± standard deviation) were 45.1 ± 5.5 mm

(length) and 5.7 ± 0.9 mm (width). The weights varied between 17 and 92 mg, with a mean of 49.3 ± 11.7 mg (N = 600). The germination rate of F. pennsylvanica was tested after 0 (control), 2, 10 and 15 days of storage in water. Every variation of the treatment was tested using three replications with

50 samaras ( Baskin and Baskin, 2001). The duration of storage in water was similar to the mean flood times (depending on the altitude) during the vegetation period in the floodplain forest investigated ( Klausnitzer and Schmidt, 2002). For the purposes of storing the samaras in water a basin was used for buy RO4929097 each treatment (diameter = 29 cm; depth = 9 cm), filled with distilled water and kept at room temperature. Distilled water was used to allow for comparability with other studies. The samaras were placed on the surface of the water, sinking over the course of the experiment. The subsequent germination test followed the ISTA (International Seed Testing Association) guidelines (ISTA, 2005) for ash, namely in a germination box on moist paper and in a germination cabinet with alternating temperatures, 16 h at 20 °C in darkness and 8 h at 30 °C in light. The same temperatures were used successfully for germination tests on F. pennsylvanica by Steinbauer (1937) and Bonner (1974). The germination test was terminated

after 20 days following the recommendation made by Baskin and Baskin (2001, p. 19). The data were analysed using Origin 8G and SPSS 11.5. Given the sample size N = 12, the critical value D0.05 was used for verification Inositol monophosphatase 1 (with N = 12: 0.375; Sachs, 1997). Significant differences were considered at the P < 0.05 level. A non-linear regression analysis was performed in order to predict the number of germinated seeds as a function of the duration of storage in water. In order to address data obtained for the different variants over time, different fitting models were compared using the χ2 minimisation fitting routine in Origin 8G. The fitting was based on 200 iterations. The Boltzmann fit was selected as the best fitting model on the basis of an evaluation of the goodness-of-fit criteria (R2 and χ2/df values).

, 1996, Namkoong, 2002, McKinnel, 2002, Bariteau, 2003 and Aravanopoulos, 2011) and much scientific attention has been paid to evolutionary and adaptive processes (e.g. Eriksson et al., 1993; Namkoong et al., 2002; Le Corre and Kremer, 2003 and Le Corre and Kremer, 2012) as a basis. However, a general application and scaling-up of the verifiers

proposed by Namkoong selleck products et al. (2002) have not yet been feasible due to the difficulties summarized above. Any relevant set of indicators for trends in genetic diversity must include components at different scales (local/landscape/national/regional/global), involving the amount of diversity and how it is distributed in space. There is a need to identify genetically appropriate indicators and, at the same time, not to inflate the already large number of indicators that exist at global and regional scales. The State–Pressure–Benefit–Response (S–P–B–R) loop developed by UNEP/CBD/AHTEG, 2011a and UNEP/CBD/AHTEG,

2011b and Sparks et al. (2011) provides a well-considered and appropriate framework to ensure that the suggested set of indicators meet the requirements of being scientifically sound, Atezolizumab supplier realistic, and policy relevant; and the framework has been adopted for implementation by BIP, 2013. The identification of indicators of tree genetic diversity should therefore preferably take place within such a framework and result in a set of S–P–B–R indicators. In Table 5 we list what we consider to be relevant operational indicators

and their type (state, pressure, benefit, response) at different geographic levels (global, regional/national and local) under the headline indicator trends in genetic diversity of tree species. Our table is not necessarily exhaustive, but proposes a fairly complete set of indicators RG7420 order and has been made in congruence with Table 2. However, no separate pressure indicators are identified. Pressure indicators of genetic diversity are intrinsically linked with state indicators and the identification of the impact of any kind of pressure will have to rely on the knowledge of the state. Response indicators are referred to as response–benefit, because the rationale for a response is typically based on benefit. In Table 5 we subdivide the headline indicator trends in genetic diversity of tree species into seven operational indicators. These are appraised based on 21 verifiable indicators using a total of 34 verifiers. Genetic diversity indicators that are proposed in order to assess the adaptive potential of forest tree species from the global to the local level present different characteristics, such as indicator classification (state, pressure, benefit, response), reference level (global, regional, national, local), type of work needed (field, lab, web-based search, etc.), feasibility and type of expertise (direct measurement, or based on experimental analysis), level of informativeness, and cost.

0 (five instances) to 0.76 with the mode in the 0.6–0.65 interval (Supplemental Fig. S1). Figure options Download full-size image Download high-quality image (354 K) Download as PowerPoint slide We selected loci that have multiple alleles in most to all of the 54 populations and are independent at the population level, i.e., that are on separate chromosomes or sufficiently far apart on the same chromosome to show minimal linkage disequilibrium. When two syntenic candidate microhaps were sufficiently close to show significant LD in several populations, we selected the locus with higher average heterozygosity in more or all of the eight major geographical regions into

which the populations cluster (see Table S1). Our development of this panel has been undertaken to demonstrate E7080 price that such a SNP-based resource can be developed and be of value in lineage/familial identification. By the very nature of these 31 multiallelic Metformin research buy loci that we have documented, proof of principle now exists. We also find the microhap loci have value for ancestry inference and individual identification. The SNP and haplotype

frequencies for the microhaps in this study are available from the authors. They are also available in the web-accessible ALFRED database (http://alfred.med.yale.edu) where they can be retrieved in a search by using the key word “microhap”. The size (molecular extent) range of the 31 microhaps is 18 bp to 201 bp with an average of 107.5 bp and a median value of 97 bp. The overall levels of heterozygosity and genotype resolvability are very good. A locus with only two alleles (e.g., a single SNP) can have heterozygosity no greater than 0.5, while a locus with three alleles can have heterozygosity of 0.667.

In general, the maximum heterozygosity occurs when all alleles have the same frequency. The median heterozygosity for these 31 loci is 0.55 for the 54 populations studied and ranges from 0.40 to 0.63. Edoxaban 26 of the 31 microhaps have heterozygosity greater than 0.5. Heterozygosity levels and genotype resolvability are also very good when examined for each of the eight major geographical regions into which the populations are grouped. The native populations of the Pacific Islands (4 populations) and the Americas (7 populations) have the lowest (but still very good) median heterozygosities of 0.53 and 0.54, respectively. Most of the 31 microhaps are on separate chromosomes or separated by molecular distances (>95 Mb) at which linkage is unlikely to exist. Eleven inter-microhap distances among syntenic loci are smaller (up to 67 Mb, cf. Table 1) and cannot be assumed to be segregating independently in families. However, the molecular extent of linkage disequilibrium (LD) varies greatly around the genome and occasionally exceeds 100 kb.

For tetracyclic triterpene glycosides, many of the methine and methylene proton signals overlapped upfield, and many of the oxygenated-methine and oxygenated-methylene proton signals of sugars overlapped in 1H-NMR spectra. Thus, one-dimensional NMR techniques were not useful for identification of those protons. To date, peak assignments in NMR data for tetracyclic triterpene glycosides have been based on previously reported data. However, many of the earlier data might be erroneous because of instrument-resolution limitations. VX-809 nmr Little NMR data are available for 20-gluco-ginsenoside Rf (4), the chemical name of

which is 6-O-[β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl]-20-O-β-D-glucopyranosyl-3β,6α,12β,20β-tetrahydroxydammar-24-ene. In this study, the definite assignment of NMR data of the compound was established for the first time by extensive NMR experiments including correlation spectroscopy, nuclear Overhauser effect spectroscopy, HSQC, and HMBC (Tables 2 and 3). By normal-phase silica gel TLC (CHCl3–MeOH–H2O = 65:35:10), Rf values were 0.27 for Re (1), 0.37 for Rf (2), 0.51 for Rg2 (3), and 0.28 for 20-gluco Rf (4). Reverse-phase ODS TLC (MeOH–H2O = 2:1) yielded Rf values of 0.57, 0.29, 0.13, and 0.65,

respectively. In 10% H2SO4 with heating, each compound was light purple on TLC. HPLC retention times were 27.1 min for Re (1), 20.6 min for Rf (2), 10.3 min for Rg2 (3), and 30.2 min for 20-gluco Rf (4). All contributing authors declare no conflicts check details of interest. This research was supported by a grant of the Next-Generation Bio-Green 21 Program (No. PJ009544) Project from the Rural Development Administration, Korea. “
“Ginseng (Panax ginseng Meyer)

is one of the most important medicinal plants and is particularly prized in Asian countries [1] and [2]. It has been a popular medicine for thousands of years in East Asia [3]. Ginseng is a deciduous perennial herb belonging to the mafosfamide family Araliaceae. Most Panax species including P. ginseng are indigenous to East Asia, but two species are found in Eastern North America [4]. Among them, P. ginseng (Korean ginseng) and Panax quinquefolius (American ginseng) have been the most widely cultivated and marketed in various commercial products because of their prominent medicinal effects, including immune system stimulation [5], anticarcinogenic activity, and reduction of blood glucose levels [6]. The two species are morphologically similar even though their origins were continentally separated by the Pacific Ocean. Most P. ginseng production is centralized in Korea and Northeast China, whereas P. quinquefolius is cultivated in China, Canada, and the United States. P. ginseng contains more than 30 kinds of triterpenoid saponin glycosides, commonly called ginsenosides, as well as other phytochemical compounds [7], [8] and [9].

Three 2 mm × 2 mm × 2 mm fragments were cut from three different segments of the right lung and fixed [2.5% glutaraldehyde and phosphate buffer 0.1 M (pH = 7.4)] for electron microscopy analysis (JEOL 1010 Transmission Electron Microscope, Tokyo, Japan).

In each electron microscopy image (50/animal), the following structural changes were analyzed: (a) shedding surface epithelium, (b) airway oedema, (c) eosinophil and neutrophil infiltration, (d) subepithelial fibrosis, (e) smooth muscle hypertrophy, (f) myofibroblast hyperplasia, (g) mucous cell hyperplasia SCR7 ic50 and (i) multinucleated cells (Antunes et al., 2010 and Abreu et al., 2011a). Pathologic findings were graded on a five-point semi-quantitative severity-based scoring system, where 0 = normal lung parenchyma, 1 = changes in 1–25%, 2 = changes in 26–50%, 3 = changes in 51–75%, and 4 = changes in 76–100% of examined tissue. Analysis was performed by two blinded pathologists. Fluorescent images of the basement membrane were obtained using a confocal microscope (Leica Microsystems Ltd., Heidelberg, Germany). Tissue sections were pretreated with PBS for 30 min and incubated overnight at room temperature in a humidified chamber with a mouse antibody against type V collagen (1:50), followed by double staining with fluorescein and rhodamine (rhodamine-conjugated goat Pembrolizumab supplier anti-mouse IgG-R, dilution 1:40, Santa Cruz Biotechnology, Santa Cruz, CA). For recipients of GFP marrow transplants,

1 week after BMDMC administration, frozen sections were treated

with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)-supplemented mounting medium Benzatropine (Vectashield, Vector Labs, Burlingame, CA), cover-slipped and examined for GFP expression by confocal microscopy. Background autofluorescence was determined through examination of 10 simultaneously prepared negative control sections that were stained only with DAPI. Images were processed and reconstructed using NIH Image software and contrast and colour levels were adjusted in Adobe Photoshop 7.0. The number of GFP+ cells per tissue area was determined by the point-counting technique (Weibel, 1990 and Araujo et al., 2010) across 10 random, non-coincident microscopic fields. Levels of interleukin (IL)-4, IL-13, transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) in lung tissue 24 h after the last challenge were evaluated by ELISA using matched antibody pairs from PrepoTech and R&D Systems (Minneapolis, MN, USA), according to manufacturer instructions. Results are expressed in pg/ml. Data were tested for normality using the Kolmogorov–Smirnov test with Lilliefors correction and the homogeneity of variances was assessed with the Levene median test. If both conditions were satisfied, two-way ANOVA, followed by Tukey’s test when required, was used for the comparison of differences among the groups. Nonparametric data were analyzed using ANOVA on ranks followed by Tukey’s test.

A very broad scope of east-west interaction among the Northeast Asian societies of this time is thus demonstrated (Zhushchikhovskaya, 2006). At higher latitudes in Northeastern China and the Russian Far East, the vast Amur River system provided Northeast Asia’s most productive interior fishery. In ethnohistoric times most of the Amur Basin’s considerable human population was aggregated into a small number of large settlements scattered along the Amur and its major Sungari and Ussuri tributaries. Most of the region’s known archeological sites and ethnographic period

settlements GW786034 clinical trial are found close together and in or near communities still occupied today. Settlement patters are topographically determined, as the seasonally flooding rivers have, over ages, created the Amur region

as a vast, low-lying alluvial plain with very little relief, where a relative few localities of higher elevation have provided the only suitable places for year-around stable human occupation for millennia (Aikens and Rhee, 1992, Aikens et al., 2009 and Chard, 1974). By the early Middle Holocene, people of the related and temporally overlapping Malyshevo and Kondon cultures (∼7000–4700 cal BP) were making pottery and collecting, fishing, and hunting along the Lower Amur River while living in sedentary and substantial semi-subterranean houses. The largest of these were about 150–180 m2 in floor area and contained Amylase interior storage pits as much as 2.5 m in diameter. To

the south in Primorye are known the somewhat earlier but comparable selleck chemicals Rudnaya Pristan (8600–8265 cal BP) and Chertovy Vorota (7650–7225 cal BP) sites, both with substantial pit houses and diverse cultural inventories. The diverse remains of mammals, birds, fishes, shellfishes, nuts, and acorns preserved in Chertovy Vorota, a dry cave site, indicate the breadth of the regional resource base. As in Korea, sites of the Russian Far East also increasingly document the presence of millets (Zhushchikhovskaya, 2006). Eastward across the Sea of Japan the Jomon people practiced patterns of subsistence and settlement similar to those just described, but there have also been found a number of impressively large Early and Middle Jomon (∼6000–5000 cal BP) sites containing both small nuclear family-sized houses and much larger rectangular buildings of public importance. It is now well-demonstrated that the flourishing and diversified Early Jomon economy of Japan also included, as previously described for the Korean Chulmun case, the management or cultivation of millets, azuki bean, soybean, and beefsteak plant (Perilla frutescens), all native plants still cultivated today ( Crawford, 1997, Crawford, 2006, Crawford, 2008, Crawford, 2011b and Lee, 2011).

It seems that pilocarpine acting centrally activates both salivary gland secretion

and vasodilation.7, 8 and 10 Because salivation depends on secretory mechanisms and on the increase in blood flow to the glands,23 reduction in salivation may occur if one or both mechanisms are affected. The activation of α2-adrenoceptor with moxonidine reduces the salivary secretion and the vasodilation induced by pilocarpine.15 and 10 Therefore, it is possible that moxonidine inhibits pilocarpine-induced salivation at least partially by reducing salivary gland blood flow. Besides Fulvestrant ic50 this, the vasoconstriction and the reduction of the blood flow to the salivary glands produced by the activation of the central α2-adrenoceptors is probably important for the sensation of dryness in the mouth by patients treated with moxonidine or the same type of drugs. In summary, the present results suggest that central cholinergic and α2-adrenergic mechanisms have opposite roles in the control of the salivary gland vascular resistance and blood flow. However, the increase in MAP, HR and mesenteric vascular resistance produced by the cholinergic activation in the forebrain is not affected by central α2-adrenoceptor activation, suggesting that different central mechanisms are activated by pilocarpine to produce the changes in the vascular resistance in different vascular beds. São Paulo State Foundation (FAPESP). None declared. Experimental protocols

were approved by the Animal Experimentation Ethics Committee of the Federal University of Sao Paulo (UNIFESP). We would like to thank also Solvay Pharma Ion Channel Ligand Library cell line and

Dr. P. Ernsberger for ADAMTS5 the donation of moxonidine. This research was supported by public funding from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Pesquisa (CNPq/PRONEX). “
“Species of the genus Candida are considered commensal yeasts frequently isolated from the oral cavity of healthy patients. 1, 2 and 3 However, these microorganisms can act as opportunistic pathogens under certain circumstances, such as impairment of salivary glands, long-term use of immunosuppressive drugs and antibiotics, denture wear, and malignancies. 4 and 5Candida albicans is the most commonly isolated species, being present in around 20–50% of the cases of oral infections. 6 Recently, infections with species other than C. albicans, notably Candida glabrata and Candida dubliniensis have been increasingly described. 7, 8 and 9C. glabrata has become the second most frequently isolated commensal yeast from the oral cavity, 2, 7 and 8 and it is responsible for 15% of mucosal lesions. 2C. dubliniensis is a recently described species of the genus Candida 10 primarily associated with oral candidiasis 11 in acquired immunodeficiency syndrome (AIDS) patients. Denture stomatitis is a common superficial infection of the palate oral mucosa that affects more than 65% of denture wearers.