02 microarray
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MICROARRAYS
Created b : rou 2
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Agatha C. MaturbongsSylvia Hanna Sirait
Eko Prasetya
Yusnaeni Yusuf
Umi Kulsum Nur Q.
Atika Okta Melisa
Nurhening
Rinaldi Rizal
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Microarray or DNA chip, Is a assembly of oligonucleotide or other DNA
probes fixed on a small, fine grid of surface A microarray used to analyze the expression
of many genes at the same time One approach is to use cDNA preparad from
mRNA or to recognize sequence variation ingenes
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Each dot in the microarray pictured here consists of anoligonucleotide fragment of a single gene bound to a glass slide. Geneexpression of two types of cells (e.g., one wild-type and one mutant)
can be compared by labeling the cDNA from each cell
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Schematic drawing of a DNA chip
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Computer Analysis
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What are the underlying microarraytechnology?
Gene expression
Gene expression is a highly complex and tightly
regulated process that allows a cell to respond
dynamically both to environmental stimuli and to itsown changing needs.
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling
Is the process, by which the contained in a section of
DNA, is transferred to a newly assembled piece of mRNA
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling
Is transcrip from DNA template, and carries coding
information to the site of protein syntethis
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling
Is the covalent linkage of a polyadenylyl moiety, to a
mRNA molecule at 3 end
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling
Is the transferred of information from RNA to DNA, or the
reverse of normal transcription
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling
Is DNA syntesiszed, from a mature RNA template in a
reaction catalyzed by enzym reverse transcriptase
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling
Is the non biological chemical synthesis of defined short
sequence of nucleic acids
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling
Hybridization is a process of combining complementary
single strand nucleic acids into a single molecule
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling It is a functional group in a molecule which with absorb
energy of a spesific wavelength and re-emit energy at adifferent wavelength
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1. Transcription2. Messenger RNA3. Polyadenylation
4. Reverse Transcription5. cDNA6. Oligonucleotide
Synthesis
7. Nucleotide acidHybridization
8. Fluorescence9. DNA labelling
Is an addition of chemical into DNA strand to make them
visualizable
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Scientists are using microarray technology totry understand fundamental aspects of
growth and development. Using microarray:
1) to analyse the mRNAs in cell, to reveal theexpression patterns of protein2) To detect genomic DNA sequence, toreveal absent or mutated genes
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1. Nucleic acid microarray, : gene expression,SNP detection, and genomic hybridization
2. Protein microarray : marker detection andpeptide array3. Tissue array: molecular biology and
immunohistochemistry4. Other molecule array : chemical array
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1. Investigating cellular states and processes2. Comparison of related species3. Diagnosis of genetic disease4. Genetic warming signs5. Precise diagnosis of disease6. Drug selection7.
Determination of gene function8. Pathogen resistance9. Following temporal variations in protein
expression
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ANALYSIS OF MICROARRAY DATA
Initial goal
To known Gene
expression
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ANALYSIS OF MICROARRAY DATA
Green spot only one targethybridize
Red spot only other target
hybridize Yellow spotboth hibrydize
Raw data from microarray experiment
Is shown by different fluorescent spot
Data from many spot on themicroarray will contribute to
the calculation of the relative
expression level of each gene
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Principal uses of chips
Genome-scale expression analysis
Differentiation
Response to environmental factors
Disease states
Effect of drugs
Detection of sequence variation
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Comparison focused on the
genes
Comparison focused on the
samples
General approach to analize
matriks gene
expression
M i ili i f diff
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+ - +
+ - - +
+ - - -
+ + -
- + -
Database(s)
1 2 3 4
Timepoints
Condition
1 2 3 4
Gene
A
B
C
E
D
0 60 120 180
Time
3 A C G G G C ATG 5
3 A C G G G C ATG 5
3 A C G G G A ATG 5
Local Alignment
Search Window
A
C B-
0
+
Measure similiarity of different
rows & columns
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Result of similiarity analize of gene
expression matrix ofthen display ed
as a chart, coloured according to the
difference in expression pattern
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Microarry Data Variablity
Microarray data are inherently highly variable- you are measuring mRNA levels
Any kind of measurement of thousands ofvalues across 2 samples will find some largedifferences due to chance (normaldistribution)
Must have replication and statistics to showthat differences are real
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Higher Level
Microarray data analysis
Clustering and pattern detection
Data mining and visualization
Controls and normalization of results Statistical validatation
Linkage between gene expression data and gene
sequence/function/metabolic pathways databases
Discovery of common sequences in co-regulated
genes
Meta-studies using data from multiple experiments
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Expression Patterns in
Different Physiological States
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How different components of cells smoothly integrate their
activity?
Alternative physiological states of an organism offer the
possibility of extracting (from an entire genome) a subset of
genes that underlie a particular life process.
Effect of the reconfiguration of the expressions patterns,
different of genes active in the two states.
In yeast : Saccharomyces cerevisiae
Diauxy.Fermentative Oxidative metabolism
In rats and fruit flies (higher organisms)
Changes in gene expressions patterns in sleep and wakefulness.
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The diauxic shift in Saccharomyces cerevisiae
Capability of adapting metabolism to a variety of
environmental conditions.
Presence of glucose :
Metabolic anaerobic
Glucose Ethanol,
via glycolysis and fermentation(Emden-Meyerhof pathway).
Exhaustion of glucose, leads diauticshift :
Metabolic aerobic
Ethanol CO2 and H2O, via theKrebs cycle and oxidativephosphorylation.
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Figure 5.3 Some metabolic pathway in yeast affected by the
diauxic shift
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The shift (different source of energy)
creates two concomitant problems :
The oxidation of ethanol doesnt
provide precursor for essential
biosynthetic pathways.
Yeast must activate pathways of
defense against oxidative stress.
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High glucose :
Mig1 (in cytoplasm) , upon dephosphorylation enters the nucleus
and binds to promotors of various genes repressor.
(lot of glucose increase ATP kinase inactive Mig1
active repression of glucose-sensitive genes)
Glucose acts as a repressor, but when
its exhausted, this repression will escapeturning on a variety of genes.
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Image captured by scanner
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Expression ratio in aerobic/anaerobic states > 2 < > 4 < Number of gene 710 1030 183 203
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Upon converting from anaerobic to aerobic metabolism,
several things occur :
a. Metabolic pathways are rerouted
b. Genes related to protein show a decrease in expression
level
c. Genes related to a number of biosynthetic pathways
show reduced expression.
d. Genes involved in defense against oxidative stress from
ROS show enhanced expression.
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The characteristics of sleep are:approximately cyclic periods of reducedconsciousness, relaxation, and quiescence.
Sleep correlate with changes in brainelectrical activity. Comparison of flies and mammals may reveal
fundamental common features disguisedwithin the individual complexities of differentspecies.
Cirelli and co worker studied gene expression
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Cirelli and co-worker studied gene expressionpatterns of rats and fruit flies in three
physiological state:
1. spontaneously awake2. spontaneously asleep
3. sleep deprived
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Cirelli and co-workers studied expressionpatterns of ~ 10000 genes of the fly.
Of these, 121 were wakefulness related and
12 were sleep related. The expression of a partly overlapping set of
130 genes was moderated by time of day: 87
were more highly expressed at 4 p.m. and 43were more highly expressed at 4 a.m.
I b th fli d t th f ti ll
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In both flies and rats, the genes preferentiallyexpressed in wakefulness and sleep fell into
several different functional categories:
In flies, gene preferentially expressed in wakingstates included those encoding proteins involvedin detoxification, gene involved in defences
against immune challenge and in lipid,carbohydrate, and protein metabolism.
Genes preferentially expressed in sleep included
the glial gene anachronism, the gene encodingthe catalytic subunit of glutamate-cysteineligase and other genes involved in lipidmetabolism.
In rat brains, a much larger number of
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In rat brains, a much larger number ofgenes than in flies had raised expressionlevels during wakefulness.
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Microarrays made possible a more systematicand thorough study for transcription patternduring stages its life.
Drossophilla melanogasterhas 4028 genes (1/3 oftotal).
86 % or 3483 are most of the genes testedchanged expression levels significantly at some
stages of life. There are 3219 genes exhibited at least a
fourfold difference between their highest andlowest levels of expression.
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The expression pattern of the eyes absentmutant, there are 33 genes and 11 genes are
already known the function in eyedifferentiation or phototransduction
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To identify pattern of gene reexpression duringdevelopment, applied a peak-binding algorithmto each genes expression profile. The result are:
36.3% showed a single major peak of expression. 40.3% showed two peaks in expression :
Early in embriogenesis and elevated pupation
Late in embriogenesis and continuing into the adultstage
23.4% showed multiple peaks in expression
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Both embrionic and pupal stage , forming and
physical activity is minimal Larval and adult stage have a more active
lifestyle but statis of anatomical form
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Blue indicated low level Yellow indicated high level
E : embryos L : Larvae P : Pupae A : Adults
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Learning and memory involve changes in thestructur and biochemistry ofnerve cell
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Fact about learning and memory:The observation that memory survives periods
of coma, during which neural activity ceases,proves this.
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Is a neural phenomenon underlying learningand memory.
Is a persistent increase in strength of asynaptic connection as a result of stimulationof the upstream cell.
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Bliss and Lomo (1973):That high-frequency stimulation of a synapse
during a finite time interval produced apersistent subsequent enhancement of thepost-synaptic response
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Learning must be regarded as a specialyzedform a development
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Change expression patterns of gene can because response to LTP
Park & co-workers: Studied genes that
change their expression pattern in responseto LTP. They studied cells from the mousedentate gyrus, a structure within the
hippocampus. The hippocampus is known to be involved inmemory formation, especially spatialmemory.
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London: metropolitancity
Taxi drivers must have an exhaustiveknowledge of the metropolitan
geography
About of optimal route betweenpoints both famous dan obscureFACT about LondonTaxi Drivers
Brain scans show that London
taxi drivers have a large
hippocampus than a controlgroup; and
The hippocampus enlarges with
time spent behind the wheel
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Following high-frequency stimulation for fourseparated 1-second intervals, total RNA wasextracted after several time intervals 30, 60,
90, and 120 minutes after stimulation and thenreverse transcribed into cDNA and hybridizedonto Affymetrix GeneChip arrays.
The chip reported 12000 genes and ESTs. Sample of unstimulated tissue provided controls.
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Within all time points, 1664 genes withstatistically significant changed expressionpatterns.
Of these
The genes identified suggest that LTP producedchanges in a variety of processes affecting cellmorphology and affects interactions amongcells and between cells and the extracellularmatrix
39% upregulated
61% downregulated
Functional Category Upregulated Downregulate
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d
The extracellular matrix and its regulation + +
Membrane protein/cell structure/adhesion mol + +
Neurosteroid hormone metabolisme - +
Cytokine/growth factor/reseptor + +
Other receptors/signalling + +
Ion channel + +
Transcription factor/regulation + +
Translation + +
Neurotransmitter receptor/neuromodulator + +
Regulation of cytoskeleton + +
Mitochondrial/energy production + +
Proteases/proteases inhibitors + +
Immunoresponsive proteins/oxidative stress... + +
Myelin-related proteins - +
Chromatine structure + -
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Transglutaminase is known to be expressed inneural tissue and appears in synapses. However,it was not known to be implicated in LTP.
Cystamine is an inhibitor of tranglutaminase andalso causes disulphide exchange productingunfolding.
Transglutaminase also helps to produce proteinaggregates in Huntingtons disease. Cystamine does ameliorate Huntingtons disease
in the mouse, but the mechanism is unclear
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Is one puzzling gene Use of a specific inhibitor of CDC25B protein
product blocked LTP.
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Genes with common time profiles Events happening at particular times
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Comparison of the distributions of homologuesin the genomes of rats, humans, Drosophila, andCandida elegans suggest that the culstering is
concerved in a evolution. The clustering may contribute to a mechanism
for common regulation of expression,
reminiscent of a bacterial operon.
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There are a significant difference between human and
chimpanzee
The set of genes that show different expression between
human and chimpanzee is rich in transcription factors. The differences in expression pattern are not uniform in
different tissues. Changes in expression pattern tend to be lower in X and
highly in Y chromosomes than in autosomes
Duplicated genes tend to show a higher divergence inexpression pattern non-duplicated genes.
The differences in expression pattern are not uniform, even
across different autosomes
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Specific Tissue Nucleotides Expressed genes
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Area of brain Function in human Function in chimpanzee if
known to differ from human
Dorsolateral prefrontalcortex
Anterior cingulated cortex
Brocas areas
Central part of cerebellum
Caudate nucleus
Pre-motor cortex
Area homologues to brocas
right hemisphere
Important for higher brain functions:working memory, conscious control ofbehavior
Autonomic functions: heart rate and bloodpressure, and cognitive functions such asreward anticipation, decision making,
empathy, and emotion
Mainly language
Coordinating complex movements such aswalking
Regulation and organization of informationsent to frontal lobes
Sensory guidance of movement ,activating proximal and trunk muscles
Not entirely clear, ome involvement incommunications without syntactic
contribution to language use
Gesture, especially control over
orofacial action, including
communicative acts
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Development of antibiotic resistance in
bacteria Childhood leukaemias
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Study in vancomycin, one of most powerfulantibiotics available for use in humans
Vancomycin is a 1,5 kDa glycopeptideantibiotic isolated from a soil bacterium inBorneo,Amycolatopsis orientalis
First used clinically in 1958
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1941 first clinical use of penicillin G 1942 appearance of penicillin-resistant Staphylococcus aureus 1950s multidrug-resistant S.aureus widespread 1956 vancomycin described 1958 first clinical use of vancomycin
1960 first clinical use of methicilin 1961 appearance of methicillin-resistant S. Aureus 1960s spread of methicillin-resistant S. Aureus 1970s Methicillin-resistant S. Aureus widespread 1988 appearance of vancomycin-resistant enterococci 1992 laboratory transfer of high-level vancomycin resistance from enterococci
to methillin-resistant S.aureus 1977 appearance of vancomycin-intermediate S. Aureus in clinical setting 2002 appearance of vancomycin-resistant S. Aureus in clinical setting
Pfeltz, R.F. & Wilkinson, B.J. (2004). The escalating challenge of vancomycin resistance in Staphylococcusaureus. Curr. Drug Targets Infect. Disord.4. 273-294
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Resistance of S. Aureusstrain
Minimum inhibitoryconcentration (MIC)
Year first appeared
Sensitive 1 g/ml -
Intermediate (VISA) 8-16 g/ml 1997
Resistant (VRSA) >32 g/ml 2002
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Mongodin et.al (2003) : compared expressionpatterns of genes in VISA strains (MIC8g/ml)with VRSA strains produced by selection
The array contained 2688 oligonucleotides The experiment were run in parallel, starting with
two different clinical VISA isolates 35 genes consistently showed increased
expression, some as high as 30-fold
16 genes consistently decreased expression Many of the change of expression levels combine
to funnel metabolites to formation of ATP
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The novel development associated withgenomics is possible to identify targets-
specific proteins, essential for pathogen, that
differ from mammalian proteins sufficientlyto suggest that drugs against these proteinswould be effective against the pathogen but
non-toxic to mammals
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Leukaemia is the uncontrolled proliferation ofany blood cell at any stage of differentiationcausing by an abnormal genetic transformation
Leukaemias can be classified according to thetype of cell that proliferating :- acute lymphoblastic leukaemia- development of second cancer, is a common
and very serious complication of acutelymphoblastic leukaemia, for.i : braintumour
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In a study using 14.500 probe sets and samples from173 patient, sets of 20-40 genes were identified thatdistinguish resistance and sensitivity to four
different drugs : prednisolone, vincristine,
asparaginase, and daunorubicin at the genetic level reveals that the activities of the
drug involve some different pathways
A set of 45 genes was found to be correlated with
resistance to all four of the drugs The majority of these genes involve transcription,
DNA repair, cell-cycle maintenance and nucleic acid
metabolism
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Microarray technology enable to compare the activities ofthousand of genes in cell types from various tissuesamples in normal and diseased cell.
Expression pattern can predict effectiveness of treatmentand guide the choice of therapy.
Identification of specific genes involves in disease cansuggest targets for drug development, for instance ; onetype of acute lymphoblastic leukaemia is associated withoverexpression of the gene FLT3 for a receptor tyrosinekinase
By the knowledge of the type of leukaemia cancer, the
phycicians can choose the treatment that will probably bemost effective for that patient
Examining patterns of gene activity can identify whichpatient will probably remain free of cancer after treatmentand which will probably relapse.
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