powerpoint presentationgenomics.unl.edu/rbc_2016/course_files/thu2.pdf · internal standards for...
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Metabolomics and Functional
Proteomics
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Major Issue with Label-free Quantitative
Metabolomics
Ion suppression
• Signal intensity of an analyte in MS depends on its concentration and
ionization efficiency. Ionization efficiency depends on other components
in the matrix
Solution:• The use of isotope coded standards
– Synthesizing the requisite 13C-coded internal standard can be difficultand expensive.
• Stable isotope coding strategy by in vivo labeling - cells grow inisotopically different media
– Method is not applicable to human and animal studies
• Stable isotope coding strategy by in vitro labeling
Department of Biochemistry
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Group Specific Internal Standard
Technology (GSIST)
• GSIST is post-biosynthetic labeling strategy
• GSIST targets specific group in molecules (e.g.primary amines or carboxylic group)
• Components from control samples andexperimental samples are derivatized with twodifferent labeling agents that are chemicallyidentical but isotopically distinct
• After mixing these derivatized components, eachcomponent from control sample serves asinternal standards for determining the relativeconcentration of the chemically identicalcomponent in experimental sample
NH2-AA + NHS-CO-CH3 CH3-CO-NH-AA
NH2-AA + NHS-CO-CD3 CD3-CO-NH-AA
Control Sample
Diseased/Expt. Sample
RPC-ESI-MS
AA from control sample labeled with light form
AA from experimental sample labeled with heavy form
m/z
Department of Biochemistry
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Challenge – Lack of Common Functional
Group
Derivatization and LC-MS conditions:
• 300-fold excess of aniline at pH 4.5.• Room temperature for 2 hours• Column: Zorbax C8• Ion pairing reagent concentration: 5 mM TBA• Mobile phase pH 5.0• Elution gradient: 5-70% ACN• ESI – negative mode
Most intermediates contains:
Aldehyde
Carboxyl
Phosphate
Department of Biochemistry
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Absolute Quantification - Labeling
Schema
Sample Standards
Combine
Light & Heavy
LC-MS
Light Isotope
Labeling (L)
Heavy Isotope
Labeling (H)
Absolute quantification
based on doublet ratio
Schema 1
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
Inte
nsity, cp
s
1
4
2
3
6
5
7
8
9
11
1210
13
1415
16
17
22
18
1921
20
23
24
27
25
26
28
29
30
32
31 33
D-ribulose-mono-aniline
250 255 260 265 270 275 280 285 290 295 300 305 310 315 320 325 330 335 340 345 350m/z, amu
0.02000.04000.06000.08000.01.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e43.0e43.2e43.4e43.6e4
Inten
sity, cou
nts
304.0592 310.0793
320 325 330 335 340 345 350 355 360 365 370 375 380 385 390 395 400 405 410 415 420m/z,amu
0.02000.04000.06000.08000.01.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e43.0e43.2e43.4e43.6e43.8e44.0e44.2e44.4e44.6e44.7e4
Inten
sity, cou
nts
379.068 391.148
355 360 365 370 375 380 385 390 395 400 405 410 415 420 425 430 435 440 445 450m/z, amu
0200400600800
100012001400160018002000220024002600280030003200340036003800400042004384
Inten
sity, cou
nts
434.222416.110
a
b c d
b/ D-ribulose 5-phosphate-momo-aniline c/ D-ribose 5-phosphate-bis-paniline d/ citrate tri-aniline
Department of Biochemistry
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Epilepsy
• Recurring unprovoked (????) seizures - uncontrolled firing of neurons
• Present in approximately 2% of the population
Causes• Genetic: the direct result of a known or presumed genetic defect(s) in
which seizures are the core symptom of the disorder; e.g., SCN1A gene has been demonstrated to be associated with Epilepsy.
• Structural/metabolic: specific structural or metabolic disorder that has been proven in various studies to be associated with increased risk of epilepsy, The cause can be acquired (e.g., trauma, stroke, infection), genetic (e.g., malformations of cortical development) or both (e.g., West syndrome).
• Unknown cause: This means that the underlying cause is yet unknown. Epilepsy might be presenting because of either some genetic defect or as a result of disorder that is not yet recognized.
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Treatment of Epilepsy
• Three main treatments
– Anticonvulsant Drugs
• Topamax, Phenobarbital, benzodiazepines, Dilantin
– Ketogenic Diet
• High fat low sugar diet
– Surgery
• Localized seizure onset zones
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Lipid & Energy Metabolism:
Ketogenic “High-Fat “ Diet
TCA
Glycolysis
PPP
AcylCoA
FA
NADPH
ATP
ATP
Lactate
Citrate
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Ongoing Studies
Mouse Model of Temporal
Lobe Epilepsy
Kcna1 -/-
Generalized
hippocampal/cortical
seizures
Approx 7 per day
Human Epileptic Brain
Tissue
• Onset
• Irritative
• Silent
Our Targets
• Energy/Lipid Metabolism
• Protein Alternation/Damage
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Metabolite Analysis
Lipids
• Global Profiles
• AcylCoA
• Fatty Acid Amides
Amino Acids
• Glu, Asp, Gln, Asn
Energy Metabolism
• ATP, ADP, AMP, cAMP, Adenosine, NADH
• TCA, PPP, Glycolysis, Lactate, Cytosolic Citrate
• NADPH, GSH/GSSG
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Amino Acids-aTRAQ
Absciex website
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Amino Acids
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Summary
• No apparent difference between onset and quiet zones
• Irritative has lower amino acid levels overall
• Signaling molecules remain constant
• Serine: important molecule associated with seizures– 3-Phosphoglycerate dehydrogenase
deficiency is a severe disorder affecting the central nervous system. Patients present with congenital microcephaly, severe psychomotor retardation, and seizures.
TCA
Glycolysis
PPP
AcylCoA
FA
NADPH
ATP
ATP
Lactate
Citrate
Serine biosynthesis
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Energy Metabolism
Human Tissue
• Tissue collection prevent ATP and other molecules determination
• Molecules are degraded
Mouse Model
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Differential and Functional
Proteomics
Transcription Translation
Protein activity
PTM Protein complex
Misfolding
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Proteomics – Experimental Approach
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Differential Proteomics
Protein ID FC O/Q p Value Acession Protein
11 1.212187 0.060913 P08559 Pyruvate dehydrogenase E1 component subunit alpha
37 1.290573 0.030006 Q8N945 PRELI domain-containing protein 2
173 1.321941 0.016407 Q6P587 Acylpyruvase FAHD1, mitochondrial
51 1.316746 0.033578 Q96JH7 Deubiquitinating protein VCIP135
52 1.34685 0.011562 A6NHL2 Tubulin alpha chain-like 3
9 2.612303 0.037366 P08729 Keratin, type II cytoskeletal 7
263 1.232938 0.040662 Q96MP5 Zinc finger SWIM domain-containing protein 3
245 0.825042 0.047108 P59768 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
73 0.690256 0.080468 Q6IQ20 N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D
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Functional Proteomics -
Protein Carbonylation
From J Proteome Res. 2010 August 6; 9(8): 3766–3780.
Lysine Modification Competition
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Sample preparation
Biotinylation of carbonylated proteins
Affinity selection with avidin
nanoRPLC-MS/MS (ion trap)
Peptide/Protein ID
Trypsin digestion
Strategy for identification of
carbonylated proteins
+ O = C - protein
N = CH - protein
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Identification of
Oxidized Amino Acids
Peroxiredoxin (TSA) Thioredoxin (TRX)
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Protein Based Strategy for Quantification
• Hydrazines react with aldehydes/ketones
– Hydrazine Cy3/Cy5
• Combine 2 samples
– Control and Sample (disease/treatment)
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2DIGE for Carbonylated
Proteins
Spot Anova (p) pI MW (kDa) Oxidation Increase Spot Anova (p) pI MW (kDa) Oxidation Increase1 0.011 7.22 25 3.3 40 0.182 8.87 46 1.7
2 0.015 6.15 32 3.1 41 0.125 7.08 60 1.6
3 0.012 6.51 23 3.0 42 0.149 8.28 22 1.6
4 0.025 6.42 16 2.6 43 0.115 7.44 22 1.6
5 0.030 6.43 16 2.0 44 0.134 9.18 22 1.6
6 0.047 6.66 16 1.9 45 0.190 7.07 59 1.6
7 0.023 6.13 22 1.8 46 0.166 6.96 58 1.5
8 0.026 5.97 33 1.7 47 0.197 4.35 14 1.5
9 0.021 7.3 25 1.7 48 0.129 8.08 20 1.5
10 0.032 6.09 25 1.5 49 0.222 8.25 32 1.5
11 0.039 5.78 25 1.5 50 0.446 9.48 54 2.3
12 0.037 5.16 88 1.5 51 0.442 7.1 17 2.2
13 0.026 5.79 56 1.4 52 0.404 5.22 1.9
14 0.088 9.18 34 2.7 53 0.326 6.85 74 1.9
15 0.060 6.4 22 2.0 54 0.314 5.64 83 1.8
16 0.067 6.24 39 1.7 55 0.459 5.64 117 1.8
17 0.076 8.94 42 1.7 56 0.303 4.96 42 1.7
18 0.068 6.86 63 1.7 57 0.315 9.24 26 1.7
19 0.099 5.88 56 1.7 58 0.293 4.83 58 1.7
20 0.083 5.55 40 1.7 59 0.370 6.68 74 1.6
21 0.073 5.73 35 1.6 60 0.301 5.65 144 1.6
22 0.081 6.81 63 1.5 61 0.433 9.33 33 1.6
23 0.071 6.62 23 1.5 62 0.284 7.16 47 1.6
24 0.187 7.1 17 3.0 63 0.268 4.33 15 1.6
25 0.148 4.96 57 2.6 64 0.282 5.63 25 1.6
26 0.223 9.26 26 2.5 65 0.430 5.49 34 1.5
27 0.112 5.95 32 2.1 66 0.273 8.87 42 1.5
28 0.104 9.01 42 2.1 67 0.295 6.19 16 1.5
29 0.201 6.75 74 2.0 68 0.281 9.26 60 1.5
30 0.190 8.95 34 2.0 69 0.591 4.92 49 2.8
31 0.230 5.59 83 1.9 70 0.866 9.48 45 1.9
32 0.197 3.9 69 1.9 71 0.567 4.08 245 1.9
33 0.121 9.29 42 1.8 72 0.549 7.51 99 1.8
34 0.131 6.85 22 1.8 73 0.687 9.46 60 1.8
35 0.252 5.64 182 1.8 74 0.846 9.52 34 1.7
36 0.185 5.85 51 1.8 75 0.918 9.48 42 1.6
37 0.237 6.47 61 1.7 76 0.581 6.39 73 1.6
38 0.203 9.29 46 1.7 77 0.887 4.58 241 1.6
39 0.126 9.02 39 1.7 78 0.544 7.27 121 1.6
Silent Zone Spot with ≥ 1.5 Fold Oxidation than Pool
Legendp-value ≤ 0.05
p-value > 0.05, ≤ 0.1
p-value > 0.1, < 0.25
p-value > 0.25, < 0.5
p-value > 0.5Legend
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Affected Proteins
• VDAC1
– Elevated levels in refractory epilepsy
– Adenine transport (ATP from mitochondria)
– Interacts with bcl-2 family proteins
– Mitochondrial permeability-CytoC
– At the plasma membrane
• Increased cellular NADH=closure
• Modulate Aβ induced cell death
• Redox homeostasis
• UCHL1
– Mutations associated with Parkinson’s Disease
– 3.2% prevalance of comorbidity
• Creatine kinase
– Pretreatment with creatine reduces PTZ induced seizures
• HSP70
– Full coverage in MASCOT
• Actin
– Low score, only 2 peptides
GAPDH
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GAPDH – Key Energy Metabolism
Enzyme
Fructose-6-phosphate / 0.49 / 2.23 / B D-6-Phospho-glucono-δ-lactone / 0.48 / 4.22 / NS
Fructose 1,6-bisphosphate / 2.88 / 2.04 / B 6-Phospho-gluconate / 0.18 / 11.85 / B
Ribulose-5-phosphate / 0.15 / 7.31 / BGlyceraldehyde-3-phosphate / 2.44 / 4.40 / B
3-Phospho-D-glycerol / 0.55 / 2.23 / B
Ribose-5-phosphate / 1.72 / 12.94 / B Xylulose-5-phosphate / 0.34 /8.37 / B
Sedoheptulose-7-phosphate / NS
Erythrose-4-phosphate / 0.20 / 3.32 / B
Glycolysis Pentose Phosphate Pathway
Dihydroxy-acetone-phosphate /
0.54 / 3.49 / B
ATPADP
NAD NADH
3-Phosphoglycerate / 1.10 / 0.86 / B
2-Phosphoglycerate / NS
Phosphoenolpyruvate / 0.13 / 0.13 / B
Pyruvate / ND
Acetyl CoA / NN
ATP
ADPATP
NADHNAD
TCA Cycle
Isocitrate / 5.46 / 2.07 / B
α-Ketoglutarate / 2.24 / 3.60 / B
Succinyl CoA / NN
Succinate / 11.46 / 2.98 / B
Fumarate / 11.46 / 2.98 / B
Malate / 2.62 / 2.28 /B
Oxaloacetate / 27.10 / 1.96 / B
FAD
NADH
NADH
ATP
ADP
NADPNADPH
NADPNADPH
Glyceraldehyd-3-phosphate / 2.44 / 4.40 / B
Fructose-6-Phosphate / 0.49 / 2.23 / B
Glyceraldehyde-3-phosphate / 2.44 / 4.40 / B
ATPADP
Glucose-6-phosphate / 1.83 / 1.98 / B
Glucose / 75.98 / 5.98 / B
ADP
NADCitrate /
58.27 / 3.72 / B
NAD
AMP / 0.29 / 0.92 / B
ATP / 2.45 / 1.77 / B
ADP / 1.01 / 1.42 / B
Conc. umol/g DW
RSD, % n=5
Quant. meth
GAPDH
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Proteomics – Impact on Energy Metabolism
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Ex Vivo Indirect Measurement
TCA
Glycolysis
PPP
AcylCoA
FA
NADPH
NADH ATP
NADH
GA
PD
H
Transketolase
G6PDH
Determine NADH and NADPH production:
Brain lysate
Excess NAD and NADP
Excess of individual substrates:
• Glucose 6 phosphate – PPP activity (NADPH)
• Ribulose 5 phosphate – PPP activity of nonoxidative branch (NADH)
• Glyceraldehyde 3 P – Glycolysis and TCA Activities (NADH)
• Pyruvate – TCA Activities (NADH)
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Potential Model: Hyper-Sensitive GAPDH ?
TCA
Glycolysis
PPP
AcylCoA
FA
Glucose
Lipids
KD Diet
ATP
NADPH
GA
PD
H
TCA
Glycolysis
PPP
AcylCoA
FA
Glucose
Lipids
KD Diet
ATP
NADPH
GA
PD
H
Pro-convulsant Anti-convulsant
Potential Treatment:
Increase Redox CapacityUse alternative sources for TCA cycle such as TCA intermediates or Acetoacetyl-CoA???
Prelim Data:
High VitC and pyruvate can reduce frequency of seizures
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PTZ induced C. elegans Model
siRNA Method• Grow HT115(DE3) with plasmid ON• Plate 500uL on NGM +Carb +IPTG
ON• Place 30 N2 worms at L1/L2 stage
– Currently synchronizing so all start L1
• Grow to adult (approx 60 hours)• Plate worms to test plate
Initial Targets• Lis1 – neuronal development/seizures
(positive control• Fgt1 – glucose transporter• GFP – negative control
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Sampling
We dreamed and schemed tobuild tools that will detectmolecules and the biologicalsignatures they bear.
While physicians dream that
these technologies can impact their world.
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Preferred Matrix for Diagnostics
and Drug Testing is Plasma not
Blood
Majority of Clinical Tests use Plasma• Clinical range usually ng/mL• Some tests require pg/mL• Latest LC/MS technology only requires a
few microliters of plasma
Plasma Prepared by Centrifugation at Lab Site
Majority of Pharma Tests use Plasma• Dosage range usually ng/mL• FDA still prefers plasma data for IND, NDA• Latest LC/MS technology only requires
one microliter of plasma
For LC/MS a majority of
the plasma sample is
often discarded
Confidential
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Spo
t Si
ze25% 50% 70%
Hematocrit
Alternative: Dried Blood
Spot Cards
Introduced 50 years ago
Basically filter paper with printed circles
Each circle requires about 25 µL of blood
Standard punch recovers 3.2 µL of blood
which translates into 1.8 µL of plasma
Performance affected by hematocrit
Hematocrit = volume % of red blood cells, typically
45% but can vary from 20 to 70%
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NoviPlex Plasma Separation
Device
Within 3 minutes, a 2.5
uL plasma aliquot is
extracted from whole
blood.
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Noviplex Tested with ELISA
Kearney et al. tested 84 patients for thepresence of antibodies to H. pylori in bloodsamples obtained by venipuncture and bythe NoviPlex card. There was a high degreeof correlation of quantitative EIA resultsbetween specimens obtained byvenipuncture as compared to thoseobtained by the NoviPlex card (r=0.98).There was also a high degree of correlationbetween duplicated cards (r=0.996). Thesensitivity and specificity of the NoviPlexdried plasma card were calculated using thevenipuncture specimen as the goldstandard. This revealed a sensitivity of 93 %and a specificity of 100 % in first duplicateand 98% in second duplicate .
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Vit. D Derivatization and Selectivity
CH 3
CH3
H
H
HO
HO
CH 3
CH3
H
H
HO
HO
N N O
ON
N+
CH3
CH2
HN N O
ON
N+
CH3
CH2
m/z = 107
m/z = 207
m/z = 149
SecoSETSecosteroid product ions
The derivative portion of the molecules
► contains a quaternary amine
► provides the dominant product ions
► provides the same mass for all vitamin D
analogues
The steroid portion provides
► unique chromatographic retention
► precursor ion masses that differ
Functional group specific derivatization along with enhanced ionization of the
derivative increases selectivity.
Secosterioid Signal Enhancing Tag
Derivatization is
based on a Diels
Alder addition
reaction
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Accuracy of Method
NIST-SRM972a L3
LLE100
NIST provided
(measured)
25OHVD2 (ng/ml) 13.650.41 13.35
25OHVD3 (ng/ml) 19.710.64 19.81
Total (ng/ml) 33.361.05 33.16
Accuracy of the LLE kit using the SecoSET derivatization reagent for quantification
was evaluated with a plasma sample (NIST-SRM972a L3) obtained from NIST
(Gaithersburg, MD). Vitamins in plasma sample were derivatized with SecoSET. The
accuracy of the LLE kit was evaluated relative to values provided by NIST and found
to have a % RSD of 0.4% in the total vitamin D concentration between the
measurement and NIST value.
LOD ~10 pg/mL depending on MS sensitivity; LOD of newest triple quads is 1 pg/mL.
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Noviplex Samples Stability
Stability of 25OHVD3 in plasma (short-term)
Weeks
0 2 4 6 8 10
% Re
lative
value(
T0=10
0%)
0
20
40
60
80
100
120
140
160
-80
-20
4
RT
37
Stability of 25OHVD3 in plasma (long-term)
Months
0 2 4 6 8 10 12 14
% Rela
tive va
lue (T0
=100%
)
0
20
40
60
80
100
120
140
160-80
-20
4
RT
37
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• Deposit blood
onto surface of
the NoviMet
card:
• Test area will
change to red in
30 sec
• Wait 3 min and
peel of the top
layer of the card
• Let collection
disc to air-dry for
15 min
• Transfer
collection disk
into a well of the
96-well plate
(with flat bottom)
• Use plastic tools
to move the disk
and place it onto
the bottom
• Add 10 µL nitric
acid (100%) and
incubate for 2 hrs
at RT
• Add 190 µL water
and incubate for 1
hr at room
temperature
• Add 200 µL
Internal Standard
(100 ppb Ga in
water)
• Inject the samples
directly into ICP-MS
• Collect data for:
Na(23), K(39), Ca(40),
Se(78)
• Other options:
Mg(24), P(31), S(34),
Mn(55), Fe(56/57),
Zn(64), As(75), Cd(111),
Hg(202)
Determination of Na, K, Ca and Se
Levels in Plasma Using NoviMet
Card
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Results1
35
–1
50
mM
NormalRange
NoviMetData
ClinicalData
14
3.3
+/-
5.3
mM
14
1 m
M
Na
3.5
–5
.5 m
M
NormalRange
NoviMetData
ClinicalData
5.5
+/-
0.2
mM
5.4
mM
K
2.1
–2
.8 m
M
NormalRange
NoviMetData
ClinicalData
2.5
+/-
0.3
mM
2.4
mM
Ca
0.8
0 –
1.6
0 µ
M
NormalRange
NoviMetData
ClinicalData
0.7
8 +
/-0
.07
µM
N/A
Se
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Thank You