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Differences in Relative Abundance of Putative Genes Involved in the Synthesis of PULCA

in E. huxleyi Cultured at 18°C and 23°C

Kristen Runge and Joe Wilson

Department of Biological Sciences, BIOL 351L-Molecular and Cell Biology Lab-Section 4

California State University, San Marcos

Abstract

Emiliania huxleyi (E. huxleyi) may someday serve a much larger role in the biofuel

industry. This marine microalgae is known to survive in a wide range of marine environments

and temperatures. E. huxleyi is one of a few microalgae that are also able to produce large

quantities of polyunsaturated long chain alkenones, or stable energy storing lipids. The aim of

this experiment was to compare Emiliania huxleyi cells cultured at 18°C and 23°C and determine

the relative abundance of the putative genes Acetyl-CoA carboxylase (ACC) and long chain fatty

acid synthase (LCFAS) involved in the synthesis of poly-unsaturated long chain fatty acids. E.

huxleyi cells were cultured under two temperature conditions: 23°C and under stress at 18°C.

RNA extraction from samples of E. huxleyi cultured at two temperature conditions, RNA gel

electrophoresis, complementary DNA synthesis, real time PCR, and melt curve analysis led to

the determination of the relative abundance of the LCFAS and ACC genes in E. huxleyi cultured

at 23°C and 18°C. It was hypothesized that the transcripts of key enzymes involved in PULCA

biosynthesis would exhibit significantly higher expression under temperature stress conditions at

18°C. It was found that the both the LCFAS gene and the ACC gene were down regulated in E.

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huxleyi cultured under 18°C temperature stress conditions as determined from the analysis of

melt curves produced from real time PCR. From these findings, it can be concluded that the

conditions under which E. huxleyi is cultured influences LCFAS and ACC gene regulation. Such

variation in the relative abundance of the regulation of the LCFAS and ACC genes are likely to

correlate with involvement in synthesis of poly-unsaturated long chain fatty acids. Further

analysis of these findings may facilitate understanding of the neutral lipid biosynthesis

pathway.3

Introduction

Renewable energy sources have been a major topic of discussion in this last turn of the

century. While solar panels, nuclear power plants and plant produced biodiesels are already in

use throughout the world, there is one biofuel source that has the potential to solve many

ecological and consumption dilemmas that have plagued renewable fuel sources. This potentially

prodigious fuel source stems from the formation of very stable lipids called polyunsaturated long

chain alkenones, or long chain hydrocarbons of 35-41 carbons in length, containing methyl ends

and 1-4 C-C double bonds, hereafter referred to in short as PULCA (O’Neil et al.,

2012)(Inagaki, 2009).

PULCA are most notedly produced in microalgae and first were most famously used as

a historic bioindicator of ocean temperature (Volkman, 1980)(O’Neil et al., 2012). PULCA are

only produced in halophytes, a group of algae named for their unique flagella. Within a sub-

group, halophytes called coccolithophores, named for their calcium carbonate disks covering its

entirety, PULCA are being studied greatly in the model organisms like Emiliania huxleyi. E.

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huxleyi are a species of marine algae that are ubiquitous in the world’s oceans and produce great

algal blooms that can be seen from space (Benthian, 2005)(Volkman, 1980)(O’Neil et al., 2012).

This organism has great potential for use in biofuel production due indirectly to these

characteristics (O’Neil et al., 2012).

The biosynthetic pathway for PULCA production is not well understood (O’Neil et al.,

2012). Considerable amounts of research need to be performed before PULCA can be mass

produced as a fuel contributor. Increased unsaturation as temperature decreases in PULCA is a

vital piece of the mystery that has been brought to light in recent years (Eltgroth, 2005) and is

associated with stressful growing conditions. In the experiment being presented here, the

comparison of the relative abundance of genes associated with the synthesis of PULCA was

made. The genes utilized in this experiment are known to produce enzymes used in the

biosynthesis of PULCA.

Analysis of gene expression was performed using real time, or quantitative, PCR

(qPCR) methods. To become available for the qPCR process, messenger RNA (mRNA) must be

isolated from cell cultures following their lysing. Said mRNA are targeted based on specific

sequencing for cDNA synthesis (Bustin, 2004). Reverse transcription, the process of producing

complete DNA (cDNA) from mRNA, utilizes the enzyme reverse transcriptase to synthesize

cDNA. Once the cDNA is synthesized, it goes through qPCR to replicate for detection. Each

cycle of cDNA replication produces greater concentrations of cDNA, which is quantified by

detecting intensity of cDNA fluorescence due to the addition of the dye SYBR Green (Bustin,

2004). During cycling, when the double stranded cDNA is produced SYBR Green attaches to it

and the qPCR machine excites the dye, causing it to emit light. The light is picked up by a

photoreceptor in the machine and quantified (Bustin, 2004). The amount of cDNA is quantified

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once the fluorescence reaches a significant intensity, called the cycle threshold.

It was hypothesized that gene expression for synthesizing enzymes associated with the

biosynthetic pathway for PULCA would be in greater abundance in cultures of E. huxleyi grown

at a stressful temperature of 18°C when compared to those grown at a control temperature of

23°C. The results of this experiment increases the understanding of the biosynthetic pathway of

PULCA in E. huxleyi which may potentially lead to a much more ecologically friendly fuel

source in the near future.

Materials and Methods

RNA Extraction from E. Huxleyi

The first steps towards analysis of gene expression of target genes involved in neutral

lipid biosynthesis in E. huxleyi was isolation and extraction of total RNA from E. huxleyi cells

grown at 18°C and 23°C. E. huxleyi cells grown at 18°C were lysed by grinding in liquid

nitrogen in order to gain access to the nucleic acid RNA. Once lysed, cells were transferred to a

sterile 50 mL centrifuge tube and resuspended in 10 mL of RNA extraction buffer (4 M

guanidium thicyanate, 25 mM sodium citrate, 0.5% sarkosyl, 0.1 M β-mercaptoethanol, pH 7.0),

then vortexed for 30 seconds. Within the extraction buffer, guanidium thicyanate was used as a

strong protein denaturant to inactivate RNAses, sarkosyl was used to; solubilize cell membranes

and to disrupt hydrophobic interactions in proteins in order to inactivate RNAses by

denaturation, and β-mercaptoethanol was added in order to reduce disulfide bonds present in

RNAses. Next, a phenol:chloroform:isoamyl alcohol extraction was performed. 1 mL of 2 M

sodium acetate (pH 4.0) was added to the 50 mL centrifuge tube then vortexed for 30 seconds,

followed by the addition of 10 mL of water-saturated phenol (pH 4.3) then voretexed for 30

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seconds, and finally the addition of 2 mL of chloroform:isoamyl alcohol (24:1) then vortexed for

30 seconds. The 50 mL centrifuge tube containing the sample was then centrifuged at room

temperature for 10 minutes at 5000 xg. The process of alcohol extraction was conducted in order

to separate proteins from nucleic acids and DNA from RNA. The RNA containing upper

aqueous phase was then removed from the 50 mL centrifuge tube and combined with an equal

volume of isopropanol in a new 50 mL centrifuge tube. Isopropanol was used to facilitate the

first of two precipitation reactions of RNA; the addition of isopropanol concentrated the RNA to

allow for its removal from the RNA extraction buffer. This sample was then mixed and

incubated at -20°C for one week; an incubation period of 1 week in comparison to 45 minutes

will significantly increase product yield, total RNA.

After a 1 week incubation period, the sample was precipitated a second time in order to

further purify RNA within the sample. The RNA extraction sample was centrifuged at 4°C for 10

minutes at 10,000 xg. This process of centrifugation pelleted the sample. The pellet was then

resuspended in 500 µL of sterile water and transferred to a microfuge tube. Resuspension of the

pellet in sterile water was done in order to dissolve the RNA into solution. Within the new

microfuge tube, an equal volume (500 µL) of 4 M LiCl was added and mixed, then placed on ice

for 45 minutes. Lithium chloride was used to facilitate the second precipitation reaction; lithium

chloride separated the RNA from any remaining contaminants. The sample was then centrifuged

for 5 minutes at full speed. After centrifugation, the pellet was washed in cold 70% ethanol, air-

dried, and resuspended in 50-100 µL of water. The pellet was washed in 70% ethanol in order to

remove any remaining lithium chloride associated with the RNA. After completion of RNA

extraction and purification, the concentration of RNA in the sample was determined via UV

spectroscopy from its absorbance at 260 nm.

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RNA Gel Electrophoresis

Next, in order to assess the integrity of the previously isolated sample of total RNA

extracted from E. huxleyi grown at 18°C, denaturing agarose gel electrophoresis was conducted.

First, a 50 mL gel at 1.5% agarose was made; 0.75 g of agarose, 36.5 of sterile water, and 5 mL

of 10X MOPS/EDTA buffer (0.2 M MOPS, 50 mM sodium acetate, 10 mM EDTA, pH 7.0)

were dissolved. The agarose was then cooled to about 55°C. 8.5 mL of 37% formaldehyde was

mixed into the agarose, then immediately poured into a gel tray and left to solidify in a fume

hood. 37% formaldehyde was added, as part of the agarose gel, to serve as an RNA denaturant

during electrophoresis; addition of formaldehyde directly to the agarose gel maintains the

denatured state of RNA during electrophoresis. Commonly used RNA denaturants for

electrophoresis are formaldehyde and glyoxal/dimethyl sulfoxide. Formaldehyde was used

because it affords a greater detection sensitivity than glyoxal/dimethyl sulfoixde. Once the gel

was solidified, it was loaded into the electrophoresis unit. The electrophoresis unit was filled

with running buffer, 1X MOPS/EDTA buffer (prepared from 10X stock of 0.2 M MOPS, 50 mM

sodium acetate, 10 mM EDTA, pH 7.0), until the gel was completely submerged. RNA samples

were then prepared prior to being loaded into the gel.

2X RNA-loading dye (62.5% formamide, 1.14 M formaldehyde, 1.25X MOPS/EDTA

Buffer, 200 µg/mL xylene cyanol, 50 µg/mL ethidium bromide) was added to the RNA sample to

bring its final volume to 1X; formamide and formaldehyde in the RNA-loading dye were used as

denaturants of the secondary structure of RNA. Then, 4 µg of total RNA, 1 µL of solution with

an RNA concentration of 1.7749 µg/µL, was combined with 2.5 µg of RNA size marker, mixed,

and heated at 65°C for 10 minutes prior to being loaded in the gel. The RNA sample was then

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loaded into a well of the gel. Gel electrophoresis was run at 75 volts until the RNA migrated

through the gel and electrophoresis was complete. The gel was then photographed for further

analysis.

cDNA Synthesis

The next step was to perform a complementary DNA (cDNA) synthesis reaction from

total RNA using oligo dT primer. cDNA is a DNA copy synthesized from mRNA. This was

completed by utilizing the enzyme used to generate cDNA, reverse transcriptase.

First, the template RNA solution was thawed on ice. Oligo dT primer (500 ng/µL), 5X

first strand synthesis buffer, DTT (100mM), dNTP mix (5 mM each dNTP) and RNase-free

water were also thawed but at room temperature. Immediately after thawing, each solution was

mixed by vortexing and briefly centrifuged in order to collect residual residue liquid from the

sides of the tube, then stored on ice. A template:primer mixture was made by combining 1 µL of

template RNA (1 µg), 11 µL of RNase-free water, and 1 µL oligo-dT primer (500 ng/µL) to

make a solution in a microfuge tube with a total volume of 13.0 µL; in solution, oligo-dT primer

had a final concentration of 25 ng/µL. Within the template:primer mixture; template RNA served

as the reverse transcription template to create cDNA, RNase-free water was used in order to

prevent unwanted cleavage of RNA, and oligo dT primer was essential in order to hybridize with

the poly-A tail of mRNA to create the necessary free 3’OH group for reverse transcriptase to

bind to in order to initiate transcription. The template:primer mixture was heated for 5 minutes at

70°C, then briefly centrifuged and placed on ice. 4.0 µL of 5X first strand synthesis buffer, 2.0

µL of dNTP mix, 1.0 µL of RT enhancer, and 1.0 µL of reverse transcriptase mix (50 U/µL) was

then added to the template:primer mixture for a total volume of 21.0 µL; in solution, 5X buffer

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had a final concentration of 1X, dNTP mix had a final concentration of 0.5 mM each dNTP, and

reverse transcriptase mix had a final concentration of 2.5 U/µL. Within this mixture, dNTP was

added in order to provide essential building blocks (nucleotide bases) for new DNA strands, RT

enhancer was added in order to enhance the overall specificity and yield of reverse transcription,

and the 5X first strand synthesis buffer was added to provide adequate salt and ionic composition

to facilitate enzyme function of reverse transcriptase.

This solution was thoroughly mixed and vortexed for 5 seconds, then briefly centrifuged to

collect residual liquid from the walls of the tube. The solution was then incubated for 50 minutes

at 42°C. An incubation period with a cool temperature allowed for the primer to bind to DNA.

Reverse transcription was initiated and conducted by reverse transcriptase during this incubation

period. Reverse transcriptase was then inactivated by another incubation period for 2 minutes at

95°C. From this, DNA was synthesized and saved for further analysis.

Real Time PCR

The next step was to quantitate the relative abundance of 3 genes of interest from the

previously synthesized DNA; Acetyl-CoA carboxylase (ACC), long chain fatty acid synthase

(LCFAS), and a “house keeping” or control gene. Regulation of gene expression was analyzed

using melt curve data produced from real-time reverse transcriptase PCR.

Triplicate reactions and a no template control were performed for real time PCR analysis.

A SYBR green master mix containing the following components was made on a per reaction

basis: 10.0 µL SYBR green super mix, 3.0 µL primer A (2 µM), 3.0 µL primer B (2 µM), and 4.0

µL of dH2O, for a total solution volume of 20 µL. SYBR green was the dye used to fluoresce the

DNA. This particular dye has proven to be useful for real time PCR because SYBR fluorescence

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increases >110-fold when it binds to double stranded DNA. Primers A and B were used due to

their specificity for the genes of interest, ACC and LCFAS. 5 µL of a 1:10 dilution of cDNA

synthesized via cDNA synthesis was then added to each of the three replicates. Each solution

was then loaded into wells within the PCR plate; a no template control was also loaded into wells

within the PCR plate for each primer set used. The PCR plate was then covered, sealed with

optical sealing tape, and placed in the real time iCycler. Real time PCR was run based on the

following parameters: polymerase activation was run at 95°C for 10 minutes for 1 cycle,

denaturation at 95°C for 10 seconds, primer annealing at 59°C for 30 seconds, and finally

extension at 72°C for 30 seconds was all run for a total of 40 cycles. Melt curves were then

produced by running the following parameters: denaturation was run at 95°C for 1 minute,

renaturation at 55°C for 1 minutes, and finally denaturation again set to ramp 0.5°C every 10

seconds for 1 cycle.

Results

RNA Concentration and Purity from RNA Extraction

From NanoDrop analysis, total RNA concentration

was observed to be 1774.9ng/µL, with a A260/A280 ratio of

1.56. This ratio is considered low in comparison to the ideal

ratio ranging from 1.8-2.1.

RNA Gel Electrophoresis

The sample of total RNA from E. huxleyi cultured

at 18°C was separated by gel electrophoresis. Two visible

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bands of rRNA were produced at approximately 5Kb and 2Kb (Figure 1). Both bands were

expressed with high and relatively equal intensity, as determined by thick, bright bands. A

mRNA smear was observed ranging from 0.7-10Kb.

Real Time PCR-Fold Change

Ideal data produced from qPCR was used for analysis; regulation of the LCFAS and ACC

genes was observed and analyzed. Both target genes of

interest were observed to be down regulated (Figure 2). The

LCFAS gene was down regulated at a fold change of -

130.99. The ACC gene was down regulated at a fold change of -41.26. The fold changes

calculated for each gene were obtained from Equation 1.

Equation 1: 2^(∆ ∆Ct(exp.-ctrl.))

∆ ∆Ct(exp.-ctrl.), represents the difference between the average threshold values for the

experimental group and the control group. These average threshold values were calculated by

subtracting the average threshold value for a reference gene from the raw threshold values,

accounting for systematic error associated with the reverse transcription and qPCR processes.

The number of cycles in which threshold amplification of cDNA was reached was determined

from amplification plots (Figure 3-A,B, & C).

Figure 1. RNA Gel Electrophoresis. Intact, target rRNA is marked at 28S and 18S. A multitude of mRNA smears the lane from around 0.7-10Kb.

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Real Time PCR-Melt Curves

The melt curves (Table 1)

were determined at the temperature

in which half of the cDNA are single

stranded. Detection of cDNA was

due to attachment of SYBR green I

to single stranded cDNA molecules.

Experimental melt temperatures

found in Table 1 represent calculated

averages from measuring the

temperature at which the curves peak

(Figure 4). Melt temperatures were determined to be very close to the expected melt

temperatures. The largest difference in melt temperatures, calculated when comparing the results

of the ACC gene, had a difference of 1.7°C. LCFAS temperature had no difference from the

actual melt temperature, at 88.5°C, and the control primer sequence had a melt temperature

difference of 0.5°C. Threshold peaks for automatic peak detection were all set to 1.00 (Table 1).

Figure 2. Comparison of the fold changes between experimental and control groups varying in E. huxleyi culture temperature. The fold changes for the gene expression of LCFAS and ACC were determined, comparing their regulation at the experimental temperature of 18°C and at the control temperature of 23°C.

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Figure 3 A,B,C. Amplification plots. A) An amplification plot of the cDNA produced in the experimental culture. The control and ACC cDNA did not reach the designated cycle threshold of 45.3. B) Amplification plot of the ideal control group. Cycle threshold was selected to be 32.8. The control primer sequence (blue), LCFAS primer sequence (violet) and the ACC primer sequence (red) all show successful readings, with noise (all other strands) not reaching the cycle threshold. C) Cycle threshold was selected to be 50.2. The control primer sequence (green), LCFAS primer sequence (blue) and the ACC primer sequence (red) al show successful readings above the cycle threshold.

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Figure 4. A melt curve plot of an ideal experimental sample. This sample contained 3 primers specific to the target

cDNA. The green curves represent the reference cDNA, the blue curves represent the LCFAS cDNA, and the red

curve represents the ACC cDNA. The solid horizontal blue line is the threshold for automatic peak detection, and

was set to 1.00. All other colored curves represent noise.

Targeted Gene Sequence

Control LCFAS ACC

Melt Temperature Avg. (°C) 88.6 88.5 93.2

Expected Melt Temp. (°C) 88 88.5 91.5

Threshold for Peak Detection 1.00 1.00 1.00

Length of Product (bp) 88 78 99

Table 1. Melt curve values of ideal experimental sample. The sample, analyzed via qPCR, contained 3 sets of

manufactured primers for the targeting of specific gene sequences.

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Discussion

The amount of total RNA isolated from E. huxleyi cells cultured at 18°C was 1774.9

ng/µL; this value quantitates the amount of total RNA isolated. Purity of isolated total RNA was

then evaluated based on the A260/A280 ratio. From isolated total RNA with a concentration of

1774.9 ng/µL, the observed A260/A280 ratio was 1.56. This value is considered low in comparison

to the ideal ratio ranging from 1.8-2.1. A A260/A280 ratio within the ideal ratio range indicates a

highly pure sample of isolated total RNA. A A260/A280 ratio lower than the ideal ratio indicates

that the collected sample likely contained other cellular components in addition to total RNA.

From this, it can be concluded that the collected sample of total RNA isolated from E. huxleyi

cells cultured at 18°C was highly concentrated with total RNA, but is not likely a highly pure

sample, as the A260/A280 ratio did not fall within the ideal ratio range.

The sample of isolated total RNA from E. huxleyi cultured at 18°C was separated by gel

electrophoresis. The development of two visible bands of RNA at approximately 5Kb and 2Kb

indicate the presence of 28S rRNA and 18S rRNA. 28S rRNA is the structural RNA for the large

ribosomal subunit of ribosomes, while 18S rRNA is the structural RNA for the small ribosomal

subunit. 28S rRNA has a known size of 5Kb and 18S rRNA has a known size of 1.9Kb.

Expression of both bands with high and relatively equal intensity indicates that rRNA was

successfully isolated and could be further analyzed.

With the sample of isolated total RNA, cDNA synthesis was conducted for further

analysis via real time PCR. From real time PCR, regulation of the target genes LCFAS and ACC

was observed and analyzed. From Equation 1, the fold change for the LCFAS gene was

calculated to be -130.99 and -41.26 for the ACC gene. The negative values for each of the

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calculated fold changes indicate down regulation of both target genes. These findings suggest

that when E. huxleyi cells are cultured at 18°C, both the LCFAS gene and the ACC gene are

decreased in expression. The lesser value of fold change of the LCFAS gene in comparison to

the ACC gene indicates that the LCFAS gene is down regulated to a greater extent than the ACC

gene. Such differences in the down regulation of the target genes of interest suggest that the

ACC gene is less down regulated and therefore more abundant in E. huxleyi cells cultured at

18°C in comparison to the LCFAS gene.

The melting curve from qPCR analysis provides a method of identification of double

stranded DNA molecules. As the cDNA molecules are heated up the bonds between single

cDNA strands disassociate. The melting curve represents these disassociations. As cDNA goes

from one strand to two strands, the SYBR I dye also dissociates from the molecule. As 50% of

the cDNA separate its strand, the lack of fluorescence is detected. This point is dependent on not

only the size of the cDNA molecule, but also the sequence and the amount of guanine-cytosine

bonds. The melting points determined from the ideal melting curve were very close to the

expected melting curves (Table 1). It was determined that the cDNA represented in the analysis

of qPCR were the same gene sequences that were targeted in this experiment.

These findings appear to indicate that E. huxleyi cultured at 18°C down regulate the

expression of the LCFAS gene and ACC gene. This contradicts the proposed hypothesis that the

transcripts of key enzymes involved in PULCA biosynthesis would exhibit significantly higher

expression under temperature stress conditions at 18°C. Rather than increased expression of the

LCFAS gene and the ACC gene, both genes were down regulated when exposed to temperature

stress conditions at 18°C. Further studies would likely need to be conducted in order to further

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evaluate the abundance and expression of the LCFAS and ACC genes in relation to the synthesis

of PULCA in E. huxleyi cells cultured at 18°C and 23.°C

Future studies of the neutral lipid biosynthesis pathway may include analysis of the

regulation of a series of different target genes within this pathway. The same procedure as this

experiment could be conducted, however primers for different target genes of interest within the

pathway would be utilized. This would allow for analysis of the regulation of a variety of

different genes within the same pathway. From this experiment, if down regulation is observed,

such findings are likely to indicate that differences in temperature conditions are a likely

influential factor in gene regulation within the neutral lipid biosynthesis pathway. Alternatively

if up regulation of the different target genes is observed, factors other than the temperature

conditions in which E. huxleyi were cultured at would likely be contributing to the regulation of

genes within the neutral lipid biosynthesis pathway.

A potential source of error may be due to the amount of stress experienced by the E.

huxleyi cells cultured at 18°C. It is possible that the E. huxleyi cells cultured at 18°C were

exposed to these low temperatures conditions for an extended duration of time. Due to these

conditions, the cells used for this experiment may have been dying or in a weakened state. In

order to correct this potential source of error, both E. huxleyi cell samples may benefit from

being cultured at 23°C first, then one of the sample’s environmental conditions could be reduced

to 18°C. This is likely to produce more accurate results for analysis.

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