![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Nutrition, The Pennsylvania State University, University Park, PA 16802;
*
Department of Biological Sciences, California State University at Chico, Chico, CA 95929; and
Department of Neuroscience and Anatomy, College of Medicine, The Pennsylvania State University, Hershey, PA 17033
2To whom correspondence should be addressed. E-mail: its{at}psu.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • iron deficiency • ferritin • brain • rats • mRNA
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
,2
) and can be viewed as both a storage pool for iron and as a site of cellular detoxification (3
,4
). In several neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease, ferritin subunit expression, and brain iron homeostasis in general, may be altered (5
). Abnormal levels of iron and perhaps altered regulation of ferritin in the presence of oxidative stress may be important factors in the pathogenesis of these disorders. Therefore, examining the regulation of ferritin expression in brain neuronal cells may provide insight into the prevention of oxidative damage.
In the brain, ferritin subunit concentration and distribution are dependent on iron status and the age and health of the animals (3
,4
,6
–9
). The levels of H and L ferritin in the brain have been examined both immunohistochemically (3
,4
,10
) and quantitatively (7
–9
) in young rats. Some researchers (7
–9
) have used dietary iron deficiency to examine regional changes in ferritin subunit levels; others (4
,9
) have measured mRNA in whole brain. A previous study (9
) showed that iron deficiency changed the H to L ferritin protein ratio in striatum and substantia nigra, the brain regions rich in iron and dopamine. A change in protein content reflects a change in gene product at levels of transcription or translation or the mRNA stability. To date, we know that ferritin protein is heterogeneously distributed across brain regions, but the mRNA expression of ferritin subunits in brain regions has not been examined fully. Therefore, a logical research question is whether iron deficiency affects ferritin subunit mRNA expression in these regions and whether the mRNA expression is correlated with its protein expression within a region.
The cell types that contain ferritin may vary with iron status, age, and the presence or absence of disease (3
,6
,11
,12
). In brain, H ferritin is found predominantly in neurons, whereas L ferritin is found in microglial cells (11
). Oligodendrocytes contain both H and L subunits, suggesting that this cell type may have a need for both storage and rapid mobilization of iron for cellular needs (11
). Neuropathology may cause a deviation from pathways for storage and metabolism of iron, resulting in a modification of cell types that either produce or contain ferritin molecules (12
). One of our research interests is to determine whether brain adaptation to iron deficiency results in alterations in which cells produce and contain the isoforms of ferritin. Given the dynamic adjustments that the brain makes in terms of iron storage and metabolism, we examined the possibility that a H ferritin–rich cell population could be an advantage to the iron-deficient brain. Our previous studies examined changes in the ferritin subunit ratio with dietary iron deficiency (9
), but did not identify either the cell types that contained ferritin subunits or the location of the ferritin subunit mRNA.
Thus, the purpose of the current study was to determine ferritin subunit mRNA expression in the brain regions of control and iron-deficient rats, and to determine whether the cell types that produce ferritin subunits are altered by dietary iron deficiency.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Male Sprague-Dawley rats (n = 26; 21 d old; Harlan Sprague Dawley, Indianapolis, IN) were randomly assigned to two dietary treatment groups: control (35 mg Fe/kg diet) or iron deficient (3.5 mg Fe/kg diet). The diets consisted of a nutritionally complete formula [AIN 93G diet (13
)] modified to contain different amounts of ferrous sulfate (Table 1
). For assessment of iron, diets were digested and analyzed for iron content on a flame atomic absorption spectrophotometer. Rats had free access to food and water 24 h/d; lights were turned off between 1900 and 0700 h. Room temperature was maintained at 25 ± 1°C. All animal procedures were approved by The Pennsylvania State University Animal Care and Use Committee.
|
Hematological variables, liver iron, plasma iron, TIBC and ferritin protein determination.
Hematological variables and TIBC were measured from blood samples as previously described (9
). Liver and plasma iron were measured by atomic absorption spectrometry (9
). H and L ferritin protein and transferrin receptor (TfR) protein contents were measured by ELISA. The antibodies used for ferritin subunit proteins were described (9
). The primary antibody for TfR was mouse anti-rat TfR monoclonal antibody (Cat. no.MCA-155, Serotec, Oxford, UK). The secondary antibody was sheep anti-mouse immunoglobulin (Ig) G alkaline phosphatase conjugate (Cat. no. A-5324, Sigma). The primary and secondary antibody concentrations used were 1:500 and 1:1000, respectively. A standard for rat TfR was not available. ELISA measurements were performed using a 96-well plate reader (Model EL340, BioTek Instruments Winooski, VT) at two wavelengths (405 and 570) nm. The methods were previously described in Han et al. (9
).
35S-dUTP–labeled in situ hybridization.
Brain sections. Sections (10 µm) were made using a cryostat and placed on poly L-lysine-coated slides. Slides were stored at -70°C for later in situ hybridization.
Probe. A 388-bp fragment of rat H ferritin cDNA (from coding region) was ligated into pBluescript Sk+/- via the KpnI and SacI sites. After amplification, the vector was cut by either EcoRI or SacI to produce antisense or sense strands. Similarly, a 489-bp fragment of rat L ferritin cDNA insert (from coding region) was ligated into PGM T easy vector via the EcoRI and NcoI sites. After amplification, the vector was cut by either EcoRI or NcoI to produce antisense or sense strands. Linear DNA was purified from 0.75% agarose gel and quantified by UV spectrophotometry. 35S-dUTP (Cat. no. NEG739H, NEN Life Sciences, Boston, MA) was incorporated into transcripts using the T3/T7 riboprobe in vitro transcription system (Cat. no. P1420, Promega, Madison, WI). Unincorporated radionucleotide was removed using a NucTrap probe purification column (Cat. no. 400701, Stratagene, La Jolla, CA). Sense strands of H and L probes were used as negative controls to make sure there was no cross hybridization.
Hybridization. Brain sections were fixed for 30 min in 4% paraformaldehyde followed by 10 min acetylation in 0.1 mol/L triethanolamine (TEA) containing 0.25% acetic acid. Slides were rinsed in ddH2O for 1 min and dehydrated through a series of ethanol dilutions for 3 min each (50, 70, 95, 100 and 100%). Denatured riboprobes (5 x 106 dpm) were added to each slide and hybridized at 50°C overnight in a humidified chamber. The next day, slides were rinsed in 4X SSC/0.02 mol/L dithiothreitol (DTT) and 4X SSC for 10 min each, then treated with RNase A (20 µg/mL RNase A in 5X Tris-EDTA buffer containing 0.5 mol/L NaCl, 0.01 mol/L Tris, 0.001 mol/L EDTA, pH 8.0) at 37°C for 30 min. Slides were subjected to a high criterion wash (50% formamide, 1X SSC, 10 mmol/L DTT) for 1 h at 55°C followed by 0.5X SSC/0.02 mol/L ß-mercaptoethanol wash for 2 h and the series of dehydrations. Slides were exposed to low energy phosphor screen (Cat. no. 63–0033-59, Amersham Biosciences, Sunnyvale, CA) and read on a phosphor imager (Storm System 860, Amersham Biosciences). The brain regions exposed to isotope were selected, and the density of mRNA was measured by Image Quant 5.1 (Amersham Biosciences).
Brain immunohistochemistry.
Iron, H and L ferritin immunohistochemistry was performed using a peroxidase/antiperoxidase method with 3',3'-diaminobenzidine (DAB) as a chromogen. The method has been described (11
). The primary antibodies for H and L ferritin were rabbit anti-rat polyclonal antibodies [L ferritin antibody was created in the laboratory, and H ferritin antibody was provided by Dr. James R. Connor (Hershey Medical School, Hershey, PA)]. The secondary antibody was anti-rabbit IgG developed in goat (Cat. no. R1131, Sigma). The tertiary antibody was peroxidase anti-peroxidase developed in rabbit (Cat. no. P2026, Sigma). The primary, secondary and tertiary antibody dilutions used were 1:500, 1:100 and 1:100, respectively, all in 30 g/L dry milk (blotto).
Digoxingenin (DIG)-labeled in situ hybridization.
DIG-labeled in situ hybridization was performed to identify the cell types containing ferritin mRNA. We used DIG instead of 35S to maintain the morphology of the cells because radioactive labeling usually destroys the cell membrane after high temperature washes.
Linear plasmids containing H and L ferritin cDNA were prepared as described in the in situ hybridization section. DIG-linked dUTP was incorporated into transcripts using a DIG RNA labeling kit (Cat. no. 1175025, Roche Molecular Biochemicals, Indianapolis, IN). Labeled probe was purified using a High Pure PCR Product Purification kit (Cat. no. 1732668, Roche Molecular Biochemicals, Indianapolis, IN). The labeling efficiency was detected using a DIG Quantification test (Cat. no. 1669958, Roche Molecular Biochemicals). Slides were fixed with 4% paraformaldehyde and washed with PBS. Sections were treated with 1 mg/L proteinase K at 37°C for 30 min and then acetylated with 0.1 mol/L TEA for 10 min. Solutions containing 5–10 ng of probe were loaded on each section, and slides were hybridized in a humidified chamber at 42°C overnight. The next day, slides were washed through 4X SSC, 2X SSC and 0.5X SSC at 37°C for 15 min each. The slides were detected using DIG Nuclei Acid Detection kit (Cat. no. 1175041, Roche Molecular Biochemicals).
Statistics.
Regression analysis was used to study the relationships among brain iron, ferritin subunit protein and ferritin mRNA contents in whole brain. Two-way ANOVA was used to study the difference in brain ferritin mRNA content between control and iron-deficient rats as well as differences between brain regions within treatment groups. Interaction terms were also considered. Normality of distributions of data were checked and data were not transformed before ANOVA. Student’s t test was used to analyze hematological variables, plasma and liver iron, dietary iron content, and TIBC. The 
-level for the analyses was set at P < 0.05. The data were analyzed using the SAS system for Windows, version 6.12 (SAS Institute, Cary, NC).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The control diet contained 41 mg Fe/kg and the iron-deficient diet contained 1.3 mg Fe/kg. In iron-deficient rats, hemoglobin, hematocrit, and plasma and liver iron were lower and TIBC was greater than in control rats (P < 0.05) (Table 2
).
|
H and L ferritin mRNA had the same locations in nearly all brain regions studied (Fig. 1
, panels A and B) The mRNA for both subunits was found in the white matter of the cerebellum, hippocampus, ventricles, medial habenular nuclei and pons. The only difference was that H ferritin mRNA was observed in the genu facial nerve in the pons, but L ferritin mRNA was not detectable in the same area. Iron deficiency did not affect the mRNA distribution of ferritin subunits in the brain (Fig. 1
, panels C and D). Hybridization using H and L ferritin sense probes showed no difference from the background.
|
). In pons, there was 
50% more mRNA (P < 0.05) in brains of iron-deficient rats compared with controls. L ferritin mRNA content was relatively constant across brain regions (Fig. 2
B), but levels were 40% lower (P < 0.05) in striatum, pons and cerebellum of iron- deficient rats compared with controls. Neither H nor L ferritin protein contents were significantly correlated with their mRNA contents in brain regions.
|
), but not between their mRNAs (P > 0.05). H and L ferritin proteins were negatively correlated with the TfR protein content in the brain (R2 = 0.3063, n = 12, H ferritin and TfR regression plot; R2 = 0.3172, n = 12, L ferritin and TfR regression plot, P < 0.05) (Fig. 3
B and C).
|
, panels A and C), whereas H ferritin protein was found only in neurons and oligodendrocytes (Fig. 4
, panel B). Iron deficiency did not affect the types of cells containing iron or ferritin subunit proteins. RBC in the blood vessels in the brains of control rats also contained iron, H and L ferritin protein (L ferritin in RBC data are not shown). In iron-deficient rats, iron and ferritin were not detectable in blood vessels.
|
). Iron deficiency did not affect the types of cells containing ferritin subunit mRNA in the brain (Fig. 5)
.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Although H and L ferritin subunits are synthesized in the same location in most brain regions, the amount of H ferritin mRNA is much greater than that of L ferritin mRNA. An earlier study (9
) showed that ferritin subunit proteins have heterogeneous distributions across brain regions and that the H to L ferritin ratio is relatively constant, regardless of iron status. Data from this study showed that ferritin subunit mRNAs also have a heterogeneous distribution across brain regions and that there is a regional effect on ferritin mRNA distribution. The lack of correlations between ferritin subunit proteins and mRNAs across brain regions suggests a post-transcriptional regulation of the ferritin gene by iron status through the iron regulatory protein (IRP)/iron response element (IRE) system (14
,15
). This post-transcriptional regulation of ferritin happens in an "opposite" way to the regulation of TfR demonstrated by the negative correlations between ferritin and TfR proteins from this study.
This study showed that iron storage in ferritin is cellular specific. We demonstrated that there are four types of cells (astrocytes, oligodendrocytes, neurons and microglia cells) containing L ferritin protein and two types of cells (neurons and oligodendrocytes) containing H ferritin protein in the brains of both control and iron-deficient rats. We also showed that iron was present in the same types of cells as L ferritin, indicating that L ferritin is more associated with the presence of iron than H ferritin. We found that ferritin subunit mRNA were detectable only in neurons. Because four types of cells contain ferritin protein but only one type of cells contains mRNA, it is likely that the site of ferritin synthesis is different from the location of ferritin protein in rat brain. The data also suggested that ferritin protein could translocate among different cell types after synthesis (16
,17
).
Iron deficiency did not induce new types of cells containing both ferritin protein and mRNA. Although neuropathologies may cause a deviation from pathways for storage and metabolism of iron and result in a modification of cell types that either produce or contain ferritin molecules (12
), we did not observe the induction of new types of cells by dietary iron deficiency. It is possible that the iron pool in the cells was not completely depleted or that the dietary iron deficiency was not severe enough to cause cellular neuropathologies. The first adaptation to dietary iron deficiency is probably at the iron-related protein levels but not at the cellular level in brain (7
,8
).
H and L ferritin mRNAs were present in neurons in pons, cerebellum, hippocampus, and caudate putamen. Neurons contain ferritin genes that produce transcripts (4
). Although some cell culture studies have shown that other cells, such as oligodendrocytes also produce the H ferritin transcript (18
,19
), the current study did not support this observation. The different findings are probably due to the use of different models (in vivo vs. in vitro) in these studies. H ferritin protein in oligodendrocytes examined in this study may be transported from neurons after its synthesis. The presence of H ferritin protein along with iron and transferrin in oligodendrocytes may be essential to support biological function in this cell type (11
,18
,19
).
Iron influences L ferritin gene transcription but not H ferritin gene expression in some brain regions such as striatum and cerebellum. Both protein and mRNA contents of L ferritin decrease in striatum when iron is low, indicating that the regional iron pool is sufficiently depleted in this region to cause the transcriptional down-regulation of the L ferritin gene. Currently, the role of iron in L ferritin gene regulation is not completely understood. One study (20
) showed that in rat liver, iron altered transcription of the L ferritin gene without affecting transcription of the H ferritin gene. Transcriptional regulation of H ferritin is relatively well understood. H ferritin transcription increases in response to monokines, hormones, stress and growth factors such as tumor necrosis factor-, interleukin-1ß, thyroid-stimulating hormones and phorbol esters (21
–23
). An ongoing hypothesis is that additional iron activates the L ferritin gene for the storage of excess iron, whereas the H ferritin gene is activated to facilitate the mobilization of iron for new protein synthesis (24
,25
).
In conclusion, we investigated ferritin subunit mRNA expression in brain regions, the association between ferritin protein and mRNA, and the cell types that produce ferritin subunits and mRNAs in the brains of control and iron-deficient rats. Ferritin mRNA content has a heterogeneous distribution across brain regions. The amount of ferritin mRNA is not correlated with ferritin protein in brain regions, indicating post-transcriptional regulation by iron status through the IRP-IRE system. We identified four types of cells that contain ferritin protein but only one type that contains the mRNA. The data suggest that the site of ferritin synthesis is different from the location of ferritin protein in rat brain. Dietary iron deficiency did not induce new types of cells containing both ferritin protein and mRNA perhaps because the adaptation to brain iron deficiency must first be controlled at the protein, not the cellular level. Overall, this study provides evidence that ferritin mRNA expression in brain regions of control and iron-deficient rats differs and implies that ferritin protein may translocate among cell types after its synthesis.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: DIG, digoxingenin; DTT, dithiothereitol; Ig, immunoglobulin; IRE, iron response element; IRP, iron regulatory protein; TEA, triethanolamine; TfR, transferrin receptor; TIBC, total iron-binding capacity.
Manuscript received 12 February 2002. Initial review completed 21 March 2002. Revision accepted 30 May 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Koeppen, A. H. (1995) The history of iron in the brain. J. Neurol. Sci. 134(Suppl.):1-9.
2. Harrison, P. M. & Arosio, P. (1996) The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta 1275:161-203.[Medline]
3. Cheepsunthorn, P., Palmer, C. & Connor, J. R. (1998) Cellular distribution of ferritin subunits in postnatal rat brain. J. Comp. Neurol. 400:73-86.[Medline]
4. Hansen, T. M., Nielsen, H., Bernth, N. & Moos, T. (1999) Expression of ferritin protein and subunit mRNAs in normal and iron deficient rat brain. Mol. Brain Res. 65:186-197.[Medline]
5. Epstein, D. & Connor, J. R. (1999) The role of iron in neurodegerative disease. Bondy, S. eds. Chemicals and Neurodegenerative Disease 1999:28-50 Prominent Press Scottsdale, AZ. .
6. Roskams, A. J. & Connor, J. R. (1994) Iron, transferrin and ferritin in the rat brain during development and aging. J. Neurochem. 63:709-716.[Medline]
7. Erikson, K. M., Pinero, D. J., Connor, J. R. & Beard, J. L. (1997) Regional brain iron, ferritin and transferrin concentrations during iron deficiency and iron repletion in developing rats. J. Nutr. 127:2030-2038.
8. Pinero, D. J., Li, N. Q., Connor, J. R. & Beard, J. L. (2000) Variations in dietary iron alter brain iron metabolism in developing rats. J. Nutr. 130:254-263.
9. Han, J., Day, J., Thompson, K., Connor, J. R. & Beard, J. L. (2000) Iron deficiency alters H- and L-ferritin expression in rat brain. Cell. Mol. Biol. 46:517-528.
10. Blissman, G, Menzies, S., Beard, J. L., Palmer, C. & Connor, J. R. (1996) The expression of ferritin subunits and iron in oligodendrocytes in neonatal porcine brains. Dev. Neurosci. 18:274-281.[Medline]
11. Connor, J. R., Boeshore, K. L., Benkovic, S. A. & Menzies, S. L. (1994) Isoforms of ferritin have a specific cellular distribution in the brain. J. Neurosci. Res. 37:461-465.[Medline]
12. Connor, J. R., Menzies, S. L., St Martin, S. M. & Mufson, E. J. (1992) A histochemical study of iron, transferrin, and ferritin in Alzheimer’s diseased brains. J. Neurosci. Res. 31:75-83.[Medline]
13. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Instiute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
14. Owen, D. & Kuhn, L. C. (1987) Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron. EMBO J. 6:1287-1293.[Medline]
15. White, K. & Munro, H. (1988) Induction of ferritin subunit synthesis by iron is regulated at both transcriptional and translational levels. J. Biol. Chem. 263:8938-8942.
16. Hulet, S. W., Powers, S. & Connor, J. R. (1999) Distribution of transferrin and ferritin binding in normal and multiple sclerotic human brains. J. Neurol. Sci. 165:48-55.[Medline]
17. Hulet, S. W., Heyliger, S. O., Powers, S. & Connor, J. R. (2000) Oligodendrocyte progenitor cells internalize ferritin via clathrin-dependent receptor mediated endocytosis. J. Neurosci. Res. 61:52-60.[Medline]
18. Qi, Y. & Dawson, G. (1994) Hypoxia specifically and reversibly induces the synthesis of ferritin in oligodendrocytes and human oligodendrogliomas. J. Neurochem. 63:1485-1490.[Medline]
19. Sanyal, B., Polak, P. E. & Szuchet, S. (1996) Differential expression of the heavy-chain ferritin gene in non-adhered and adhered oligodendrocytes. J. Neurosci. Res. 46:187-197.[Medline]
20. Rothenberger, S., Mullner, E. W. & Kuhn, L. C. (1990) The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution. Nucleic Acids Res. 18:1175-1179.
21. Torti, S. V., Kwak, E. L., Miller, S. C., Miller, L. L., Ringold, G. M., Myambo, K. B., Young, A. P. & Torti, F. M. (1988) The molecular cloning and characterization of murine ferritin heavy chain, a tumor necrosis factor-inducible gene. J. Biol. Chem. 263:126-144.
22. Rogers, J. T., Bridges, K. R., Durmowicz, G. P., Glass, J, Auron, P. E. & Munro, H. N. (1990) Translational control during the acute phase response. Ferritin synthesis in response to interleukin-1. J. Biol. Chem. 265:4572-4578.
23. Leedman, P. J., Stein, A. R., Chin, W. W. & Rogers, J. T. (1996) Thyroid hormone modulates the interaction between iron regulatory proteins and the ferritin mRNA iron-responsive element. J. Biol. Chem. 271:12017-12023.
24. Beaumont, C., Dugast, I., Reanudie, F., Souroujon, M. & Grandchamp, B. (1989) Transcriptional regulation of ferritin H and L subunits in adult erythroid and liver cells from the mouse. J. Biol. Chem. 264:7498-7504.
25. Ponka, P. (1999) Cellular iron metabolism. Kidney Int. 55(Suppl.):S2-S11.
This article has been cited by other articles:
|
|
 |
|
  P. Hahn, Y. Qian, T. Dentchev, L. Chen, J. Beard, Z. L. Harris, and J. L. Dunaief Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration PNAS, September 21, 2004; 101(38): 13850 - 13855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kordas and R. J. Stoltzfus New Evidence of Iron and Zinc Interplay at the Enterocyte and Neural Tissues J. Nutr., June 1, 2004; 134(6): 1295 - 1298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. N. J. Strube, J. L. Beard, and A. C. Ross Iron Deficiency and Marginal Vitamin A Deficiency Affect Growth, Hematological Indices and the Regulation of Iron Metabolism Genes in Rats J. Nutr., December 1, 2002; 132(12): 3607 - 3615. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
