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Genetics, Vol. 172, 1699-1710, March 2006, Copyright © 2006
doi:10.1534/genetics.105.053389
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Department of Genetics and Developmental Biology, School of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030
2 Corresponding author: Department of Molecular Biology, Cell Biology and Biochemistry, Division of Biology and Medicine, Brown University, Laboratories for Molecular Medicine, 70 Ship St., Room 407, Providence, RI 02903.
E-mail: stephen_helfand{at}brown.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The electron transport chain in the mitochondria uses the energy from high-energy electrons released during oxidative metabolism to establish a proton gradient across the inner mitochondrial membrane. In aerobic organisms, the vast majority of ATP is produced in the mitochondria via ATP synthase, which captures the energy of the protons as they diffuse across the membrane and uses it to synthesize ATP (SARASTE 1999). UCPs generate a "proton leak" that allows the flow of protons across the mitochondrial membrane without the generation of ATP. Uncoupling due to UCPs is a ubiquitous process that occurs in all eukaryotic cells examined to date and can account for up to 20–30% of resting metabolic state (BRAND 2000).
UCPs were originally discovered as heat dissipation catalyzers involved in the thermogenic capacity of brown adipose tissue (NICHOLLS and LOCKE 1984). However, the finding of homologs of the initial UCP (later renamed UCP1) has raised questions about the in vivo physiological roles of these proteins. The mammalian UCP family includes UCP1, UCP2, UCP3, UCP4, and UCP5/brain mitochondrial carrier protein 1 (BMCP1). While UCP1 expression is restricted to brown adipose tissue, the expression of the other homologs has been shown to be specific to other tissues and their function is not restricted to thermogenesis (KRAUSS et al. 2005). UCP overexpression and loss-of-function studies have demonstrated the importance of these proteins in metabolism. UCP2-deficient mice were shown to have higher islet ATP levels, which in turn increased glucose-stimulated insulin secretion in pancreatic ß-cells (ZHANG et al. 2001). Overexpression of UCP3 led to hyperphagic mice that were lean and exhibited fat mass reduction and increased glucose clearance rates (CLAPHAM et al. 2000). Additional studies have pointed to a role of uncoupling in maintaining an adequate redox balance, which in turn would protect against the formation of free radicals during oxidative metabolism (ARSENIJEVIC et al. 2000; ECHTAY et al. 2000, 2002).
UCP5, which was the last homolog of the UCP family to be identified, shares 
35–40% amino acid identity with the other UCPs (SANCHIS et al. 1998). UCP5 expression was found to be highly enriched in the mammalian nervous system (SANCHIS et al. 1998; YU et al. 2000) and in the brain expression has been determined to be almost exclusively neuronal (KIM-HAN et al. 2001). Because nutritional status and temperature affect ucp5 transcript levels in the brain and in the liver of mice, UCP5 has been postulated to be involved in mediating metabolic adaptations (YU et al. 2000). On the other hand, on the basis of their observation that UCP5 overexpression in neuronal cell lines decreases the levels of reactive oxygen species in mitochondria, KIM-HAN et al. (2001) hypothesized that UCP5 plays an important role in regulating mitochondrial oxidant production by controlling mitochondrial respiratory efficiency.
Sequence homology analysis revealed the existence of four putative UCPs in Drosophila—UCP4a, UCP4b, UCP4c, and UCP5 (JEZEK 2002)—that share 60–70% homology with their mammalian counterparts. Drosophila UCP5 uncoupling activity has been functionally characterized in the heterologous yeast system, where UCP5 expression reduces mitochondrial membrane potential and increases respiration rate. UCP5 action is governed by the mechanisms known to regulate the UCPs characterized to date, including fatty acid stimulation and GDP inhibition (FRIDELL et al. 2004). ucp5 is expressed throughout Drosophila development but at higher levels in adults, where it is expressed most abundantly in the head (FRIDELL et al. 2004).
Although UCPs have been shown to be involved in multiple pathways related to metabolism, the biological role of the predominantly brain-expressed UCP5 is not understood. It is possible that due to its brain expression, UCP5 plays a systemic role in fine tuning organismal energy homeostasis. Alternatively, UCP5 may be involved in specific metabolic tasks in the neuronal tissues where its expression is restricted. Using a UCP5 knockout (UCP5KO) line, we sought to investigate the in vivo involvement of UCP5 mitochondrial uncoupling in metabolism and aging.
On the basis of the assumption that mitochondria in UCP5KO flies would produce more ATP per molecule of oxygen consumed during respiration, we predicted that UCP5KO flies would be more resistant to food deprivation and would gain more weight than controls. Surprisingly, UCP5KO flies were highly sensitive to starvation stress and gained less weight than controls on high-calorie diets. Moreover, UCP5KO flies exhibited a decreased level of fertility and lived longer than controls on low-calorie diets. The results show that UCP5 may play an important role in Drosophila metabolic homeostasis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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2,3) allele or the TM6 balancer. Heterozygous BmcpBG02446 males were crossed to double balancer TM3/TM6B females. Following this cross, homozygous excision lines were established from individual males that had lost the mini-white marker. Additionally, homozygous control lines were established from individual males resulting from the BmcpBG02446/TM6 x TM3/TM6B cross.
To create transgenic ucp5 lines, the full-length ucp5 cDNA fragment fused at the 3'-end to the FLAG epitope tag (FT) was subcloned from the pRS426 vector (FRIDELL et al. 2004) into the pUAST transformation vector. The pUAST–UCP5::FT construct was injected into the germline of w; 2,3/TM3 flies. Independent homozygous UCP5::FT transgenic lines were established and backcrossed to w1118 flies for 10 generations. To generate a UCP5::FT line that was homozygous UCP5KO, a UCP5::FT transgenic line containing the ucp5 transgene on the second chromosome was crossed to w;Sco/+;TM3/+ flies to obtain w;UCP5::FT/Sco;TM3/+ flies. In parallel, w;CyO/+;UCP5KO/TM6B males were generated and crossed to w;UCP5::FT/Sco;TM3/+ females to ultimately obtain homozygous w;UCP5::FT/ UCP5::FT;UCP5KO/UCP5KO flies. Generation of Elav-GAL4 driver flies that were homozygous UCP5KO was accomplished by first producing Elav-GAL4 flies that were TM3/TM6B double balanced. From the progeny of the cross of UCP5KO males with Elav-GAL4;+;TM3/TM6B females, we selected Elav-GAL4;+;UCP5KO/TM3 males, which in turn were crossed to Elav-GAL4;+;TM3/TM6B females. From the resulting progeny, we generated homozygous Elav-GAL4;+;UCP5KO/UCP5KO flies.
Drosophila maintenance:
All flies were reared on standard cornmeal agar medium (ASHBURNER 1989). Flies were passed to fresh vials every 4–6 days and maintained in humidified temperature-controlled environmental chambers at 25° throughout development. Adult flies were sorted and collected under CO2 anesthesia and allowed to recover for at least 48 hr prior to assays.
Life-span assays:
Groups of 25 newly eclosed males and 25 newly eclosed females were placed together in each vial with a total of 10–12 vials per assay. Flies were transferred to fresh vials containing the different percentage of yeast–sucrose (Y–S) diet under study every other day, and the number of dead flies was scored. Flies were maintained at 25° with 60% humidity on a 12 hr:12 hr light:dark cycle. Different yeast–sucrose calorie diets were prepared as described (MAGWERE et al. 2004). Alternate low- and high-calorie diets were implemented using standard cornmeal agar medium with or without yeast added, respectively (ASHBURNER 1989). All life-span studies were performed using 450–600 flies per assay.
Fertility analysis:
Ten to 20 newly eclosed flies were maintained in single pairs in vials containing the different percentages of yeast–sucrose diet. Flies were transferred to fresh vials every day, and the number of eggs was counted. Flies were maintained under standard conditions (see above).
Longitudinal weight analysis:
Three groups of 30–40 newly eclosed males and females were collected and weighed to determine their initial body weight. Every 3–4 days, each group of flies was anesthetized under CO2 and weighed using a Mettler Toledo AB54-S precision scale. The average weight of the flies remaining alive was then calculated. Flies were maintained under standard conditions (see above).
Starvation stress assay:
Flies were segregated by sex and groups of 25 newly eclosed flies were maintained on standard food for 10 days prior to assays. For starvation stress assays, flies were transferred to vials containing 2% agar. The number of dead flies in each vial was scored every 6–12 hr. All starvation assays were performed using 100–200 flies per line. Flies were maintained under standard conditions (see above).
Reverse transcription–PCR:
Total RNA was isolated using TRIzol reagent (Invitrogen, San Diego) and subsequent reverse transcription (RT)–PCR experiments were performed as described (FRIDELL et al. 2004). Forward and reverse primers for ucp5 amplification were 5'-ATACGAGGGCGTTCGTGG-3' and 5'-GTACTTCTTTAGTTGTTCGTA-3'. Primers for the amplification of rp49 were the same as described previously (RADYUK et al. 2003).
Genomic PCR:
Fly genomic DNA preparations were performed as described (ASHBURNER 1989). Approximately 200 ng of genomic DNA preparations was used for PCR experiments following the manufacturer's recommendations. All reagents were purchased from Invitrogen. Primers used were 7314-X (5'-GCGATGGAATCCCAATAAAACTGC-3'), 7314-Y (5'-TGACCTTGGATTTGGAGGCG-3'), and EP-d (5'-CAATCATATCGCTGTCTCACTCA-3'). Primers 7314-1 and 7314-FN are the forward and reverse primers used in the RT–PCR experiment, respectively.
Mitochondrial respiration:
Heads and thoraxes of 7- to 14-day-old flies, which had been maintained on standard food upon eclosion, were dissected on ice and kept chilled for <30 min before mitochondria isolation was performed as described (FRIDELL et al. 2005). Following measurement of total mitochondrial protein, respiration of freshly isolated mitochondria was determined in a Clark-type oxygen electrode at 25° (Hansatech, Norfolk, United Kingdom). Mitochondria were suspended at a protein concentration of 150 µg/ml in electrode buffer containing 20 mM 
-glycero-3-phosphate as substrate as described (MIWA and BRAND 2003). ADP (1 mM), oligomycin (1 µg/ml), and GDP (0.5 mM) were added sequentially to record state 3, state 4, and GDP-sensitive mitochondrial respiration, respectively. The substrate -glycero-3-phosphate, ADP, and GDP were dissolved in water, and oligomycin was dissolved in ethanol before adding to reactions. All chemicals were purchased from Sigma (St. Louis).
ATP measurements:
Steady-state ATP content was measured using the sensitive luciferin–luciferase system (MANFREDI et al. 2002). The principle of this assay is based on the fact that luciferase requires ATP for light production using luciferin as substrate. Total cellular extracts from heads and thoraxes were prepared using the guanidine hydrochloride method to prevent ATP degradation (SCHWARZE et al. 1998). Homogenates were added to reaction buffer containing luciferin and assayed using a TD-20/20 luminometer (Turner Designs).
Sugars, glycogen, and triglyceride body composition determination:
Fly homogenates were prepared as described (CLARK and KEITH 1988). Groups of 10 flies were anesthetized under CO2 and weighed prior to homogenization in 1 ml of chilled homogenization buffer (0.01 M KH2PO4, 1 mM EDTA pH 7.4). Homogenates were spun using a refrigerated microcentrifuge for 2 min at 2000 rpm and the supernatant was recovered. Triplicates with 25 µl of homogenate for each sample were aliquoted into 96-well titer plates. Total glucose was measured using the 510-A glucose determination kit (Sigma). Total amount of trehalose was measured by determining total amount of glucose after addition of 0.2 units/ml of trehalase (Sigma) for 1 hr. Glycogen determination was performed by incubating homogenate samples with 0.1 units/ml of amyloglucosidase as described (CLARK and KEITH 1988) and measuring the total amount of glucose. Glycogen composition was calculated by subtracting the total glucose composition without amyloglucosidase digestion from the total glucose composition after amyloglucosidase digestion. Triglyceride measurements were performed using the triacylglycerol hydrolysis kit 335-UV (Sigma). All results were normalized with fly weight.
Statistical analysis:
Statistical analyses for life spans were performed using a log-rank test (StatView). Differences for body composition assays were analyzed using a paired Student's t-test.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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30- to 50-bp insertion made up of a small duplication of ucp5 sequence and P-element sequence or a partial P-element truncation between the AT and the G of the translational start codon. The sequence of each of the imprecise excision lines is predicted to prevent the expression of any UCP5 protein. Precise excision of the P element in the ucp5 gene restored starvation resistance in the flies. However, imprecise excision of the P element produced flies that retained the starvation sensitivity as observed by the 42.9 and 36.2% difference in median lethality for males and females, respectively, with respect to the precise excision controls (Figure 2, E and F). Similarly, control lines that had not been exposed to the transposase allele retained the P element and the starvation sensitivity as expected (data not shown). Taken together, these results demonstrate that loss of normal UCP5 function leads to flies that are highly sensitive to nutrient deprivation.
Ectopic neuronal expression of UCP5 rescues starvation resistance:
ucp5 transcript levels are higher in the head of adult flies than in the rest of the body (FRIDELL et al. 2004). Therefore, UCP5 might function in the nervous system and contribute to the regulation of metabolic homeostasis. To test this hypothesis, we generated ucp5 transgenic flies to ectopically express UCP5 in the starvation-sensitive UCP5KO flies. An Elav-GAL4 nervous system driver line, as well as a transgenic ucp5 line, were crossed into the homozygous UCP5KO genetic background and starvation sensitivity was examined. The presence of the P-element insertion disrupting the ucp5 gene was confirmed in the newly generated lines by genomic PCR (Figure 1C). Homozygous UCP5KO flies containing the Elav-GAL4 driver element (Figure 3) or the ucp5 transgene alone (data not shown), but not both, exhibited sensitivity to starvation as compared to their matched genetic controls. However, homozygous UCP5KO flies containing the Elav-GAL4 driver element and the ucp5 transgene restored flies to almost a wild-type starvation -resistant phenotype (Figure 3). In conjunction with the genetic studies above, these transgenic studies confirm that disruption of the ucp5 gene is important in the normal response to starvation. Furthermore, the fact that restricting expression to the nervous system is sufficient to restore the starvation resistance to near-normal levels supports the hypothesis that UCP5 normally functions in the nervous system and may contribute to the regulation of metabolic homeostasis.
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UCP5KO flies live longer than controls on low-calorie diets but not on high-calorie diets:
Aging and metabolism are tightly linked. An example illustrating the linkage between aging and metabolism is the recurrent phenotype of life-span extension across species upon limiting dietary calorie intake (GUARENTE and PICARD 2005). Because UCP activity influences ATP production and metabolism, it is reasonable to expect that modulation of UCP activity will have an impact on longevity and consequently on aging. To test this prediction, life-span assays were performed using UCP5KO and control flies on diets ranging from severely calorie restricted to high-calorie content. UCP5KO flies had 10–30% shorter median life spans than controls on two different types of severely calorie-restricted diets: 1 and 1.5% Y–S. These results were expected since, as described above, UCP5KO flies are highly sensitive to food deprivation. Interestingly, upon addition of slightly more calories to their diet, 2% Y–S, UCP5KO flies had up to 18% longer median life spans than controls (Figure 4A and Table 1). Furthermore, on the still low-calorie 5% Y–S food, UCP5KO flies had a >30% increase in median life span as compared to controls. This phenotype suggests that UCP5KO flies are not merely sickly flies but instead may have an altered metabolic homeostasis. Gradual dietary calorie increases showed that UCP5KO flies live longer than controls on low-calorie diets (2 and 5% Y–S) but the same as controls on moderately higher (10% Y–S) or high (15% Y–S) calorie diets (Figure 4, B and C, and Table 1). Additional life spans were tested on alternative low- and high-calorie food diets, yielding very similar results (data not shown). Altogether, these results suggest that the highly starvation-sensitive UCP5KO flies have an altered metabolic homeostasis since, depending on the calorie intake, they can starve much faster than controls, live longer than controls on low-calorie content diets, or live for the same time as controls on high-calorie diets.
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UCP5KO flies have lower sugar levels and upon starvation use their triglyceride reserves faster than controls:
UCP5 expression is impaired in UCP5KO flies, yet these animals are comparable to those of controls in overall mitochondrial uncoupling and total levels of ATP. Nonetheless, UCP5KO flies exhibit a metabolic homeostasis imbalance as manifested by the phenotypes described above. Therefore, a possibility to consider is that UCP5 acts in small subsets of cells and that these cells are important in mediating the homeostatic control of metabolism. Mammalian UCP2 has been shown to negatively influence insulin secretion (ZHANG et al. 2001). Thus, expression of UCP2 in a very limited subset of cells, pancreatic ß-cells, can greatly affect the endocrine control of metabolic homeostasis. In flies, the secretion of insulin-like peptides and adipokinetic hormone (the insect equivalent of glucagon) is controlled by clusters of neurosecretory cells in the brain (RULIFSON et al. 2002; KIM and RULIFSON 2004). Because Drosophila UCP5 expression has also been reported to be predominantly in the nervous system, a hormonal imbalance caused by less uncoupled neurosecretory cells could explain the observed phenotypes in the UCP5KO flies. To test this possibility, we compared the level of glucose and trehalose (a fundamental sugar for the fly) in control and UCP5KO flies fed normally or starved. We detected 21% lower levels of total sugars in normally fed UCP5KO flies as compared to two ucp5 wild-type controls, W-10 and w1118. We then subjected flies to starvation stress and observed that the total level of sugars remained at their respective baselines up to 18 hr before they started to slowly decrease (Figure 8A). In parallel to the sugar measurements, we analyzed the rate of use of the two major energetic reserves, glycogen and triglyceride (TAG). These two different metabolites were stored at the same level in fed UCP5KO and control flies. Once flies are starved, glycogen and TAG reserves decreased rapidly. However, while glycogen was depleted in all fly types during the first 18 hr of starvation, UCP5KO flies consumed their TAG reserves at a much faster rate than controls (Figure 8, B and C). These results are consistent with the possibility that ucp5 mutant flies are hypoglycemic and that, upon starvation, for them to maintain their basal metabolism, they have to utilize their TAG reserves at a faster rate than controls. Then if starvation conditions persist, UCP5KO flies would exhaust their reserves faster than controls and consequently also would die faster than controls.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Recently, our laboratory functionally characterized UCP5 as a bona fide fly UCP, establishing Drosophila as an alternative model for in vivo UCP studies (FRIDELL et al. 2004). We used flies lacking UCP5 activity to determine the biological significance of UCP5 in fly metabolism. Our findings suggest an important role for UCP5 in maintaining metabolic homeostasis. We hypothesize that UCP5 influences hormonal control of metabolism.
Flies lacking UCP5 expression display an unexpected phenotype:
Since UCPs diminish the amount of ATP that can be generated from oxidative metabolism, we predicted that flies that lack UCP5 expression would perform better than controls when assayed in tests that reflect energy production. Therefore, we expected flies lacking UCP5 to be more resistant than controls to starvation stress conditions, to gain more weight than controls, and to increase their fertility over controls. However, when we compared the performance of flies that lacked UCP5 expression with genetically matched controls, we observed that UCP5KO flies died much faster when subjected to food deprivation conditions, gained less weight on high-calorie diets, and had a diminished fertility status. Previous reports have shown that the expression level of some UCPs are upregulated upon starvation (DULLOO et al. 2001; CARROLL and PORTER 2004). This suggests that uncoupling activity might be an important element in the normal starvation response. However, when we starved flies, we did not observe any significant changes in the level of ucp5 transcription (data not shown), although we cannot rule out an increase in the translation of UCP5 protein.
Lack of UCP5 expression and metabolic homeostasis alteration:
One possibility to explain the unexpected phenotypes observed in UCP5KO flies is that they become sickly because they lack UCP5 function. Therefore, the performance of sickly UCP5KO flies on any assay that involves energy production and use would be poorer than that of controls. However, when we examined the life span of flies that lacked UCP5 expression, we noted that UCP5KO flies lived considerably longer than controls under low-calorie conditions, demonstrating that UCP5KO flies may not be merely sickly flies. We also showed that ectopic neuronal expression of the ucp5 transgene was sufficient to rescue the starvation sensitivity phenotype of flies that lack normal UCP5 expression, suggesting the importance of UCP5 function in the nervous system. Since UCP5 function appears important in the nervous system, it is possible that UCP5 may play a regulatory role in the nervous system of the fly and that lack of UCP5 function leads to flies with altered metabolic homeostasis that are not able to respond to nutritional metabolic challenges.
Involvement of UCP5 in metabolic homeostasis:
As mentioned above, expression of UCP5 in the nervous system is sufficient to restore normal levels of starvation resistance, which supports the hypothesis that UCP5 function in the nervous system contributes to the regulation of metabolic homeostasis. Interestingly, the fly nervous system has specific subsets of neurosecretory cells that regulate metabolic balance. These neurosecretory cells have been compared to a "fly pancreas" in that they regulate the release of insulin-like peptides (ILPs) and adipokinetic hormone (AKH), the insect equivalent of glucagon (RULIFSON et al. 2002; KIM and RULIFSON 2004). Since flies without UCP5 have lower-than-normal levels of body sugars, it is possible that UCP5 may affect ILP and/or AKH neurosecretory cells, altering the normal metabolic balance of Drosophila. Effects on insulin levels have been observed in mice in which UCP2 activity has been shown to influence pancreatic ß-cell glucose-stimulated insulin secretion by affecting ATP/ADP ratios (ZHANG et al. 2001). Increased UCP2 activities decrease the ATP/ADP ratio in ß-cells and negatively influence insulin secretion by impeding the closure of 
-dependent channels. Moreover, similar to our results, UCP2 –/– mice displayed lower blood glucose levels and gained less weight than controls when fed high-fat diets (JOSEPH et al. 2002). Recent studies in flies showed that AKH-producing cells express the subunits that form the -dependent channel homolog, making these cells functionally similar to mammalian islet cells in the sensing and regulation of glucose homeostasis (KIM and RULIFSON 2004). We hypothesize that the loss of UCP5 activity in fly ILP and/or AKH neurosecretory cells creates a change in the ATP/ADP ratio, which is responsible for an unusual hormonal response that leads to flies with altered metabolic homeostasis. Because changes in UCP5 expression lead to lower sugar levels and altered metabolic homeostasis in the fly, further investigation on UCP5 function may provide new insights into the molecular causes underlying diabetes and other metabolic syndromes.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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