P11 - Southern California Institute for Research and Education

Report
Filippo Martini
Intern
VA Long Beach Healthcare System
Chris Reist, MD, MBA, ACOS
Program Director, Associate Chief of Staff (Research & Development)
5901 East 7th Street, Long Beach, CA 90822 (562) 826-8000 Ext. 5801
University of Bologna
Dr. Laura Mercolini
Mentor and Professor
Laboratory of Pharmaco-Toxicological Analysis; Department of
Pharmacy & Biotechnology (FaBiT), Alma Mater Studiorum University of Bologna,
Via Belmeloro 6, 40126 Bologna, Italy.
Mental Health Research Internship
Program
VA Long Beach Healthcare System
Chris Reist, MD, MBA, ACOS
Associate Chief of Staff (Research & Development)
5901 East 7th Street, Long Beach, CA 90822 (562) 826-8000 Ext. 5801
Program Director
Clinical Study Coordinator
Laboratory Project Coordinator
Chris Reist
Stephanie Alley
Neil Hoa
Acknowledgments
Martin Jadus, Ph.D. Molecular Cancer Immunology Laboratory
Amrita Ahluwalia, Ph.D. Gastroenterology and Biochemistry Laboratory
Lisheng Ge, Ph.D. Diagnostic molecular scientist
Program Objectives
The goal of the mental health internship program is
the identification, characterization and validation of
Biomarkers / Bio-signature for the foremost
mental disorders (i.e. Major Depression - MD,
Posttraumatic stress disorder - PTSD), that would
facilitate:
1. correct prediction of mental health disease risk,
2. therapeutic responses, and
3. ultimately lead to information-based diagnosis
for preventive and treatment plans of mental
health illness..
Clinical Research Trials
Observe, experience and understand the mental health
clinical research trial processes, techniques, safety
and security and privacy requirements
 Report write up
 Create Power Points and present
1) Enhancing Exposure Therapy for PostTraumatic Stress Disorder (PTSD): Virtual
Reality and Imaginal Exposure With a
Cognitive Enhancer.
2) VA Augmentation or Switching Treatment for
Improving Depression Outcomes (VAST-D)
Clinical Research Trials
Comments:
 Observed but were not able to participate
due to strict Federal Regulation law Health
Insurance Portability and Accountability Act
of 1996 (HIPAA). Only individuals whose
names are on the approved Clinical trial
study are allowed to have direct involvement
(i.e. interviews, collect, evaluate, with the
patients.
 I was only able to review the clinical trials
write up
Biomarker Laboratory Research
Hands on operation of scientific instruments and
performed Experiments used in the lab for
detection and interpretation of research data
Methods used in Cancer biomarker research Laboratory
Include:
1) Cell Culture
2) Sample Collection
and Preparation
3) mRNA purification
4) real time PCR,
5) Western blot
6) Flow Cytometry
7) ELISA
Biomarker Laboratory Research
Presentation
 Correlative Clinical Studies & Development of
Biomarkers from the Acute Effects of
Psychotropic Drug Using Cancer Cells Model
 Presentation on Nov 25, 2014 can be access on
www.scire-lb.org
Research Activities
 Attended two Lecturer seminars, and
 Presented 3 presentations
 Discuss the strategies for developing test for
mental disorders, from the bench to bedside
approaches and applications for detecting,
treating, and preventing mental health
disorders
Summary
 Gave three presentations: two Mental Health
Clinical Research Trial and one Biomarkers
Research Laboratory Presentation
 Attended Two Distinguish Lecturer Series
 Perform Pub med medical database subject
search and reference
 Contributed to two manuscripts
Manuscript
Fascin-1 knock-down of human glioma cells
reduces their microvilli/filopodia while
improving their susceptibility to lymphocytemediated cytotoxicity Neil T. Hoa1, Lisheng Ge1,
Kate L. Erickson2, Carol A. Kruse2, Andrew N.
Cornforth3, Yurii Kuznetsov4, Alex McPherson4,
Filippo Martini1,7, Martin R. Jadus1,5,6,*
1Research
Health Care Group, Veterans Affairs Medical Center Long Beach, CA 9082, USA;
2Department of Neurosurgery, David Geffen School of Medicine, University of California,
Los Angeles, Los Angeles, CA 90095, USA; 3California Stem Cells, Inc. 18301 Von Karman
Avenue, Irvine, CA 92612, USA; 4Molecular Biology and Biochemistry, University of
California, Irvine, Irvine, CA 92697, USA; 5Pathology and Laboratory Medicine Service,
Veterans Affairs Medical Center, Long Beach, CA 90822, USA; 6Department of Pathology
and Laboratory Medicine, University of California, Irvine. Orange, CA 92868, USA;
7Laboratory of Pharmaco-Toxicological Analysis; Department of Pharmacy &
Biotechnology (FaBiT), Alma Mater Studiorum - University of Bologna, Via
Belmeloro 6, 40126 Bologna, Italy.
Manuscript
Temozolomide induces the expression of the glioma Big Potassium
(gBK) ion channel, while inhibiting fascin-1 expression: A possible
synergistic target for glioma immunotherapy.
Neil T. Hoa1, Lisheng Ge1, Filippo Martini 1, 2, Vincent Chau1, Amrita Ahluwalia1, Carol
A. Kruse3, and Martin R. Jadus 1,4,5,6
1.
2.
3.
4.
5.
6.
VA Long Beach Healthcare System, Research Health Care Group, Veterans Affairs
Medical Center, Long Beach, CA 90822.
Laboratory of Pharmaco-Toxicological Analysis; Department of Pharmacy &
Biotechnology (FaBiT), Alma Mater Studiorum - University of Bologna, Via
Belmeloro 6, 40126 Bologna, Italy.
Department of Neurosurgery, David Geffen School of Medicine, University of California,
Los Angeles, Los Angeles, CA 90095.
Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Long
Beach, CA 90822.
Department of Pathology and Laboratory Medicine, University of California, Irvine.
Orange, CA 92868.
Chao Comprehensive Cancer Center, University of California, Irvine. Orange, CA 92868.
Manuscript in preparation for submission to Neuro Oncology for
December 15, 2014
Filippo Martini
Dr. Laura Mercolini
Professor & Mentor
Laboratory of Pharmaco-Toxicological Analysis;
Department of Pharmacy & Biotechnology (FaBiT),
Alma Mater Studiorum - University of Bologna,
Via Belmeloro 6, 40126 Bologna, Italy.
Presentation Outline
Mental health illness and Cancer Relationship:
Overview
II. Psychotropic drugs
III. Biomarkers/Targets

To Define Biomarker

To Design appropriated studies

Roles of biomarkers in phase I, II, and III studies
IV. Laboratory methods used in biomarker research

Sample types

Gene Analysis
 Real time-PCR (mRNA)
 Protein Analysis
 Flow Cytometry
 ELISA Assay
 Western Blot
V. Conclusion
I.
Introduction
 Psychiatric diseases are chronic, recurrent and have
a complex etiology and particularly vulnerable to the
environmental factors (i.e. stress)
 Very little understanding of the underlying disease
process
 Have very few animal models that have the validity
to predict clinical efficacy
 The effects of psychiatric drugs on the Biochemical
Modulation of Target, Target Pathway, and the
Biological/Cellular Response of the target are not
well studied
Introduction
 The current treatment of Psychiatric diseases , such
as depression, and post-traumatic stress disorder
(PTSD) is established based on a patient’s clinical
history, mental status examination, duration of
symptoms, and clinician administered symptom
checklists or patient self-reports. (not a reliable
means of measures)
 Unlike cancer management, markers and laboratory
tests are not available for the detection, diagnosis,
prevention and treatment of Psychiatric diseases .
Clinical Trials Diagram
Mechanism of Drugs effect on biological markers/targets that
correlated with effective treatment outcomes, a correlative
studies.
Psychotropic Drugs
• Aripiprazole
• Bupropion-SR
• Sertraline
The Drugs
Psychotropic Drugs
• Aripiprazole (Abilify): A second generation, or "atypical"
antipsychotics developed in the 1990’s and used to treat
schizophrenia and schizophrenia-related disorders. Side
effects: weight gain, metabolic disease, diabetes, and high
cholesterol.
Antidepressants work to balance neurotransmitters such
as serotonin, norepinephrine, and dopamine in the brain.
Have less side effects.
• Sertraline (Zoloft) is AKA selective serotonin reuptake
inhibitors (SSRIs) specific to serotonin. (The happy pill) 
• Bupropion-SR is serotonin and norepinephrine reuptake
inhibitors (SNRIs).
Mental Health
Serious mental illnesses
include Major Depression,
schizophrenia, bipolar
disorder, obsessive
compulsive disorder
(OCD), panic disorder,
posttraumatic stress
disorder (PTSD) and
borderline personality
disorder. Early detection
Slower Death
Cancer
Glioblastomas (GBM)
are tumors (AKA
glioma) that arise from
astrocytes—the starshaped cells that make
up the “glue-like,” or
supportive tissue of the
brain. Late detection
Quick Death
Similar Causes
Stress
Immune System
MENTAL Health
CANCER
Stress
Chronic Inflammation
PTSD
Major Depressive
Disorder
Mental Illness Risk
Factors:
Cancer Risk
Factors:
Genetics, aging, tobacco,
sun exposure, radiation
exposure, chemicals and
other substances, some
viruses and bacteria,
certain hormones, family
history of cancer,
alcohol, poor diet, lack of
physical activity, or being
overweight.
Genetics (parent or
sibling); Exposure to
viruses, toxins, drugs or
alcohol during pregnancy.
Overweight, Stressful life
situations, (financial, death,
divorce) A chronic medical
condition, such as cancer.
Traumatic brain injury
(TBI), such as Traumatic
experiences, (PTSD).Use of
illegal substances
Stress Perception
A PHYSIOLOGICAL
RESPONSE
CANCER
Morimoto R I Genes Dev. 2008;22:1427-1438
Copyright © 2008, Cold Spring Harbor Laboratory Press
Induced Cellular Insulin Resistance –
An In-vitro Depression Model
Palmitic Acidinduced insulin
resistance in
astrocytes to
study targets
effected by the
Drugs using cell
model
What is a Biomarker/Target?
 Biomarker - A characteristic that is objectively
measured and evaluated as an indicator of
normal biologic processes, pathogenic processes,
or pharmacologic responses to a therapeutic
intervention
Biomarkers Definitions working Group National Institutes of Health 2001
 Assay - A method for determining the presence
or quantity of a component (PCR, ELISA, Western
Blot, Flow Cytometry)
 Test - A procedure that makes use of an assay for
a particular purpose
Good biomarkers ≠ Good Assays ≠ Tests
The Biomarkers development process
Phase 1
Diagnosis
Therapeutic Drug
Monitoring
P11
BLOOD
SERUM
Depression
Prediagnosis
•Confirmation
•Staging
•Subtyping
Pretreatment
•Prognostic
•Predictive
Treatment
Sertraline
Metabolites
Intratreatment
Phase 2
•Risk
•Screening
•Early
detection
Types of Clinical Biomarkers
• Early
response or
futility
• Toxicity
monitoring
C
E
L
L
S
Posttreatment
gBK
?
•Early
endpoint
•Recurrence/
progression
monitoring
Glioma Large conductance calcium-activated potassium
channels (gBK) a possible target for Psychiatric Drug
Effects – Laboratory methods used in cancer research
for assay development for biomarker of psychiatric
disorders
Why gBK? Because it is a candidate for brain cancer
biomarker
1. Study of postmortem brain in suicide individual suffered
from major depression shows low level of BK large
conductance calcium-activated potassium channel
expression compare to normal.
2.
Neurochem Res. 2014 May;39(5):901-10. doi: 10.1007/s11064-014-12871. Epub 2014 Mar 26. Large conductance calcium-activated
potassium channels: their expression and modulation of
glutamate release from nerve terminals isolated from rat
trigeminal caudal nucleus and cerebral cortex. Samengo I1, Currò D,
Barrese V, Taglialatela M, Martire M.
Modeling Depression with
Insulin Resistance
Palmitic Acid-induced
Insulin in Glioma Cell
Line U251 and in-vitro
model
This will simulate cells in
the depressed individuals
NORMAL
CANCER
Insulin Resistance + Cancer
Proinflammatory Cytokines (IL-6 and TNF-α)
BK channels in both the neural circuitry and
endocrine output of the HPA axis
Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing
ACTH from the anterior pituitary gland and Glucocorticoid from the
adrenal cortex. Stress also activates the sympathetic nervous system,
evoking adrenaline release from the adrenal medulla. Large-
conductance calcium- and voltage-activated potassium
(BK) channels have been implicated in regulation of cellular
excitability in these systems. ………………. These results support an
important role for BK channels in both the neural circuitry and endocrine
output of the HPA axis and indicate that the stress
hyporesponsiveness in BK(-/-) mice primarily results from reduced
activation of hypothalamic PVN neurosecretory neurons.
gBK is BK
SI-S4
 Glioma large conductance
calcium-activated
potassium channels (gBK)
is highly expressed in
cancer cells and function
for ionic balance and
modulate the release of
neurotransmitters.
 gBK form has the extra
P11
three putative sites which
allow more interactions
and increase
responsiveness
?
S5-S6
The p11 protein is linked with the transport of neurotransmitters.
Found in the brain of humans and other mammals, it has been
implicated in the regulation of mood. In addition, due to its
interaction with serotonin-signaling proteins and its correlation
with symptoms of mood disorders, p11 is a new potential target for
drug therapy.
P11
gBK
Pathways by which P11 mediates
behavioral responses to
antidepressants diagram
Non-Insulin Resistance model
Methods Used in the Laboratory
Materials:
 Human Glioma Cell
line U-251
Methods:
 Aripiprazole
 Cell Culture
 Bupropion-SR
 RT-PCR
 Sertraline
 Palmitic Acid
 TNF-α antibody
 P11 (AKA: S100A10)
antibody
 gBK Antibody
 IL-6 antibody
 FACS/Flow
Cytometry
 Western Blot
 ELISA
Biological Safety
Cabinet: Personal
Protection and prevent
contamination of Cells
Culture
Cell Culture
Liquid Nitrogen
Cryogenic storage at
very cold temperatures
is presumed to provide
an indefinite longevity to
cells
Methods: Cell Culture
Images courtesy of Judy Shon- 2012 Summer Scholar
 Purpose = grow cells outside of
U251
body
 Initially cells are attached in
T75 flask
 Media: DMEM (Dulbecco
Modified Eagle Medium)
Conditions:
Insulin Resistance (IR)
inductions 24hrs with
Palmitic Acid then add the
drugs 24 hrs, harvest cells
for Assays.
IR-Control
Bupropion (300µM)
Aripiprazole (10µM)
Sertraline (5µM)
Real time - Polymerase Chain Reaction (Rt-PCR)
Method #1: Polymerase Chain Reaction (PCR)
By enabling the use of
DNA Polymerase on the
DNA sample, the
polymerase chain reaction
technique synthesizes new
complementary DNA
strands and amplifies the
sample up to millions of
times.
Real-Time qRT-PCR
 Real-Time quantitative Reverse Transcription (qRT)
polymerase chain reaction (PCR) enables detection and
measurement of products generated during each cycle of PCR
process
 Introduction of an oligonucleotide probe which was designed
to hybridize within the target sequence.
The Primers were synthesized by Operon Biotech
(Germantown, MD). The sequences were:
 gBK Forward : 5'-CGTTGGGAAGAACATTGTTC -3';
 gBK Reverse: 5'- AACTGGCTCGGTCACAAG -3';
The relative quantification of expression of the gene was
determined by 2-ΔCT
Polymerase Chain Reaction (PCR)
1. ISOLATION of mRNA
2. QUANTIFICATION of mRNA
3. cDNA Synthesis
Preparation sample for
PCR run
Polymerase Chain Reaction (PCR)
 Denaturation of the DNA
template (no fluo), SYBR
Green molecules are free in
the reaction mix (95°C for 1015 minutes)
 Primers anneal and SYBR
Green molecules bind to the
single stranded cDNA
 DNA polymerase elongates
the template and more SYBR
Green molecules bind to the
product formed resulting in
exponential increase in the
fluorescence level
Polymerase Chain Reaction (PCR)
RELATIVE QUANTIFICATION of mRNA
• Compared to the values of threshold cycle, ie the number of cycles
required so that the fluorescence signal may become significant
compared to the background.
• The genic expression quantification is assessed by the fold change
method( 2-∆∆CT )
• Using an endogenous control (18s) is possible to normalize the
amount of starting RNA, ie means to calculate ∆CT for each
sample: ∆CT= Ct target gene - Ct endogenous control
• It’s necessary to define a calibrator, ie a control sample (no treated)
that will be used to compare the other samples; this is equivalent to
calculating the ∆∆CT= ∆CT sample - ∆CT calibrator
• The relative quantification RQ = 2-∆∆CT ; this parameter tells us
how many times the gene is more expressed in the sample with
respect to the endogenous control
Polymerase Chain Reaction (PCR)
NORMAL
INSULIN RESISTANCE
10
9
8
7
6
• In normal environment
the cells didn’t show a
such as relevant
changing of gBK
expression
5
4
3
2
1
0
Aripiprazolo
Buproprion
Sertraline
• Meanwhile a significant
increase in the
expression of GBK cells
insulin resistant if
normalized with the
normal environment
Methods: Flow Cytometry/FACS Analysis
•
Flow Cytometry is a rapid way to
measure the characteristics of
individual cells. Hematopoietic
cells (blood, bone marrow
aspirate, lymph nodes, etc.)
•
Routinely used in the diagnosis of health
disorders, we used it in the lab to
analyze protein expression
•
Cell Quest Pro takes the readings from
the machine and forms it into numerical
data and graphs. Readings use 10,000
cells each
1x106 cells per 100 μl
sample ice cold
Staining Cells for
FACS Analysis
Wash with PBS 0.2% Tween 20
3 times centrifuge 400 g for 5
min
Incubate with fluorochrome
conjugated primary antibody (direct
method)
15 to 45 min 4°C in the dark 0.1-10 μg
antibody/ml
Fix: (optional) Add 100 μl fixative (4% PFA), Wash in PBS 0.2% Tween 20 3 times
centrifuge 400 g for 5 minute, Resuspend to 500 μl PBS, Store at 4°C until analysis
(within 24hr)
Flow Cytometry – how it works
Cells flow single file in a stream
of fluid
Measures multiple
characteristics of each cell
10,000 cells are analyzed
per reading
Results: gBK Flow Cytometry Analysis
In glioma cell line, insulin resistance U251 has lower
expression of gBK compared to normal U251 baseline
expression. Treatment of Psychotropic Drugs increases
gBK expression.
Aripiprazole
U251 = human glioma Cell
IR = Insulin Resistance
A = Aripiprazole (10µM)
B = Bupropion (300µM)
S = Sertraline (5µM)
Bupropion
Sertraline
Results: P11 Flow Cytometry Analysis
In glioma cell line, insulin resistance (IR) U251 has
lower expression of P11 than normal U251 baseline
expression. Treatment of Psychotropic Drugs increases
P11 expression.
U251 = human glioma Cell
IR = Insulin Resistance
A = Aripiprazole (10µM)
B = Bupropion (300µM)
S = Sertraline (5µM)
P11
P11
P11
P11
Results: TNF-α Flow Cytometry Analysis
Glioma insulin resistance (IR) cell line U251 has higher
expression of TNF-α compared to normal U251 baseline
expression. Treatment of Psychotropic Drugs decreases the
pro-inflammation cytokine TNF-α.
U251 = human glioma Cell
IR = Insulin Resistance
A = Aripiprazole (10µM)
B = Bupropion (300µM)
S = Sertraline (5µM)
TNF-α
TNF-α
TNF-α
TNF-α
Results: IL-6 Flow Cytometry Analysis
Glioma insulin resistance (IR) U251 expresses higher level IL-6
compared to normal U251. Over all the treatment of (IR) U251
with Psychotropic Drugs decreases the pro-inflammation
cytokine IL-6. Sertraline significantly reduces IL-6 level compare
to Aripiprazole and Bupropion.
U251 = human glioma Cell
IR = Insulin Resistance
A = Aripiprazole (10µM)
B = Bupropion (300µM)
S = Sertraline (5µM)
IL-6
IL-6
IL-6
IL-6
Western Blot
SDS-PAGE
Pore Size
 Gel percentage
% Gel concentration
 Proteins (-) run towards
the anode (+).
 Separated in pores by
size
http://www.siumed.edu/~bbartholomew/course_material/protein_methods.htm
http://bjpsbiotech.edublogs.org/labs-activities/western-blot/
GE Handbook: Western Blotting Principles and Methods
Western Blot
SDS –PAGE
Transfer to
membrane
Incubate with
antibody
Detect antibody
http://www.bio.davidson.edu/Courses/genomics/method/Westernblot.html
Amrita Ahluwalia, Ph.D.
Gastrointestinal(GI) Research
Results: Western Blot
Effect of Psychotropic Drugs on gBK protein
expression in insulin resistance (IR) glioma cell
line U251.
120 kDa
gBK
Β-Actin
ELISA: Enzyme-Linked ImmunoSorbent Assay
a widely used
method for
determining the
presence or absence
of a specific target
protein and its
respective
quantification.
NOVOstar- plate reader for
detection of ELISA Assays
Samples graph of Standard Curve for
known amount protein concentration
ELISA
(1) Plate is coated with a capture antibody;
(2) sample is added, and any antigen
present binds to capture antibody;
(3) detecting antibody is added, and binds
to antigen;
(4) enzyme-linked secondary antibody is
added, and binds to detecting antibody;
(5) substrate is added, and is converted by
enzyme to detectable form.
ELISA APPLICATIONS
 Determination of serum antibody
concentrations in a virus test
 home pregnancy test
 food industry when detecting potential food
allergens
 toxicology as a rapid presumptive screen for
certain classes of drugs
 various of areas such as Immunology,
Biological Pharmacy, Diagnostic industry,
and so on.
0.05
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
0.00
-0.01 0
y = 8E-05x - 0.0026
R² = 0.9889
200
400
600
IL-6 (ng/ml) + SD
OD Value
ELISA: Results
600
IR-U251
500
400
*
300
*
200
*
100
0
Control
IL-6(ng/ml)
Aripiprazole Bupropion
(10µM)
(300µM)
Sertraline
(5µM)
y = 9E-05x + 0.0126
R² = 0.9994
0.07
0.06
OD Value
TNF-α (ng/ml) + SD
Treatment
0.05
0.04
0.03
0.02
0.01
0
0
200
400
TNF-α (ng/ml)
600
350
IR-U251
300
250
200
*
150
100
*
50
*
0
Control
Aripiprazole Bupropion
(10µM)
(300µM)
Sertraline
(5µM)
P11 (ng/ml) + SD
ELISA: Results
3500
2500
IR-U251
*
*
*
1500
500
Control Aripiprazole Bupropion Sertraline
(10µM)
(300µM)
(5µM)
Treatment
Putting it all together
Pathways by which P11 and gBK mediates
behavioral responses to antidepressants
IL-6
TNF-α
P11
gBK
protein p11 (also known as S100A10)
CELL MIGRATION
AND
PROLIFERATION
Conclusion
The data from FACS Analysis, RT-PCR, Western
Blot, and ELISA suggests the mechanism of the
ACUTE (24-28 hours) effects of psychotropic drugs
(Aripiprazole (10µM), Bupropion (300µM), and
Sertraline (5µM) increase the expression of both P11
and gBK, and down regulate the pro-inflammation
cytokines IL-6 and TNF-alpha in induced insulin
resistance glioma cell line (U251) in-vitro.
gBK and P11 may be a possible biomarker candidate
use for mental illness clinical trial.
Markers and Endpoints for Trials of
Molecularly Targeted Agents
Changes in protein
function/phosphorylation
Biochemical Modulation
of Target Pathway
Protein
phosphorylation/function
Expression array
Markers of
Biological/Cellular
Response
Proliferation, Cell cycle phase,
Apoptosis, Angiogenesis,
Metabolism
Clinical Response
Objective Behavior Response
Prolonged Mental Stability
Clinical Benefit
Happier, better quality of life
Phase 3
Biochemical Modulation
of Target
Phase 2
Blood and tissue levels
Phase 1
Pharmacokinetics
Acknowledgments
I sincerely thank Dr. Reist for his support of the Mental
Health Research Internship Program, and Miss Stephanie
Alley for her patience in teaching me the mental health
clinical trial procedures, principles, assessment, and treatment
for my report.
I thank Dr. Ge for teaching me Real Time PCR and molecular
biology techniques, Dr. Amrita Ahluwalia for showing me the
western blot technique, and Dr. Jadus for using his laboratory
and his expertise in inflammation and cancer immunology.
Note
Questions and Issues
Note
Questions and Issues

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