University of Bologna Dr. Laura Mercolini Mentor and Professor Laboratory of Pharmaco-Toxicological Analysis; Department of Pharmacy & Biotechnology (FaBiT),

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. 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 Filippo Martini Intern

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 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 Post- Traumatic 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 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 www.scire-lb.orgPresentation

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 lymphocyte- mediated cytotoxicity Neil T. Hoa 1, Lisheng Ge 1, Kate L. Erickson 2, Carol A. Kruse 2, Andrew N. Cornforth 3, Yurii Kuznetsov 4, Alex McPherson 4, Filippo Martini 1,7, Martin R. Jadus 1,5,6,* 1 Research Health Care Group, Veterans Affairs Medical Center Long Beach, CA 9082, USA; 2 Department of Neurosurgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; 3 California Stem Cells, Inc. 18301 Von Karman Avenue, Irvine, CA 92612, USA; 4 Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA; 5 Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Long Beach, CA 90822, USA; 6 Department of Pathology and Laboratory Medicine, University of California, Irvine. Orange, CA 92868, USA; 7 Laboratory of Pharmaco-Toxicological Analysis; Department of Pharmacy & Biotechnology (FaBiT), Alma Mater Studiorum - University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy. The tracking number for this manuscript is AJTR0003859 NOVEMBER 14, 2014

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. Hoa 1, Lisheng Ge 1, Filippo Martini 1, 2, Vincent Chau 1, Amrita Ahluwalia 1, Carol A. Kruse 3, and Martin R. Jadus 1,4,5,6 1. VA Long Beach Healthcare System, Research Health Care Group, Veterans Affairs Medical Center, Long Beach, CA 90822. 2. Laboratory of Pharmaco-Toxicological Analysis; Department of Pharmacy & Biotechnology (FaBiT), Alma Mater Studiorum - University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy. 3. Department of Neurosurgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095. 4. Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, Long Beach, CA 90822. 5. Department of Pathology and Laboratory Medicine, University of California, Irvine. Orange, CA 92868. 6. 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 I. 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

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 patients clinical history symptom checklists patient self-reports. The current treatment of Psychiatric diseases, such as depression, and post-traumatic stress disorder (PTSD) is established based on a patients 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 Psychotropic Drugs AripiprazoleAripiprazole Bupropion-SRBupropion-SR SertralineSertraline Mechanism of Drugs effect on biological markers/targets that correlated with effective treatment outcomes, a correlative studies.

The Drugs Psychotropic Drugs Aripiprazole (Abilify): A second generation, or "atypical" antipsychotics developed in the 1990s and used to treat schizophrenia and schizophrenia-related disorders. Side effects: weight gain, metabolic disease, diabetes, and high cholesterol.Aripiprazole (Abilify): A second generation, or "atypical" antipsychotics developed in the 1990s 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)Sertraline (Zoloft) is AKA selective serotonin reuptake inhibitors (SSRIs) specific to serotonin. (The happy pill) Bupropion-SR is serotonin and norepinephrine reuptake inhibitors (SNRIs).Bupropion-SR is serotonin and norepinephrine reuptake inhibitors (SNRIs).

Cancer Glioblastomas (GBM) are tumors (AKA glioma) that arise from astrocytesthe star- shaped cells that make up the glue-like, or supportive tissue of the brain. Late detection Quick Death 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

Similar Causes CANCER Immune System Metabolism Inflammatory Response Stress MENTAL Health

Chronic Inflammation Major Depressive Disorder Stress PTSD

Mental Illness Risk Factors: 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 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.

Stress Perception Stress A PHYSIOLOGICAL RESPONSE Chronic Inflammation

Induced Cellular Insulin Resistance An In-vitro Depression Model Palmitic Acid- induced 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

Types of Clinical Biomarkers Pre- diagnosis Pre- treatment Intra- treatment Post- treatment RiskRisk ScreeningScreening Early detectionEarly detection Early response or futilityEarly response or futility Toxicity monitoringToxicity monitoring ConfirmationConfirmation StagingStaging SubtypingSubtyping Treatment Diagnosis Therapeutic Drug Monitoring P11 BLOOD SERUM gBK CELLSCELLS Phase 1 Phase 2 PrognosticPrognostic PredictivePredictive Early endpointEarly endpoint Recurrence/ progression monitoringRecurrence/ progression monitoring Depression Sertraline Metabolites ?

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/s11064-014-1287- 1. 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, Curr 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

NORMALCANCERInsulin Resistance + Cancer Proinflammatory Cytokines (IL-6 and TNF-)

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. BK channels in both the neural circuitry and endocrine output of the HPA axis

gBK is BK 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 three putative sites which allow more interactions and increase responsiveness SI-S4 S5-S6 P11 ?

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: Cell Culture RT-PCR FACS/Flow Cytometry Western Blot ELISA Methods Used in the Laboratory Materials: Human Glioma Cell line U-251 Aripiprazole Bupropion-SR Sertraline Palmitic Acid TNF- antibody P11 (AKA: S100A10) antibody gBK Antibody IL-6 antibody

Cell Culture Biological Safety Cabinet: Personal Protection and prevent contamination of Cells Culture Liquid Nitrogen Cryogenic storage at very cold temperatures is presumed to provide an indefinite longevity to cells

Purpose = grow cells outside of 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. Images courtesy of Judy Shon- 2012 Summer Scholar Methods: Cell Culture Aripiprazole (10M) Bupropion (300M)Sertraline (5M) U251 IR-Control

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-CT

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 (95C for 10- 15 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 Its 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)

In normal environment the cells didnt show a such as relevant changing of gBK expression Meanwhile a significant increase in the expression of GBK cells insulin resistant if normalized with the normal environment Polymerase Chain Reaction (PCR)

Flow Cytometry is a rapid way to measure the characteristics of individual cells. Hematopoietic cells (blood, bone marrow aspirate, lymph nodes, etc.) Methods: Flow Cytometry/FACS Analysis 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 l sample ice cold 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 4C in the dark 0.1-10 g antibody/ml Fix: (optional) Add 100 l fixative (4% PFA), Wash in PBS 0.2% Tween 20 3 times centrifuge 400 g for 5 minute, Resuspend to 500 l PBS, Store at 4C until analysis (within 24hr) Staining Cells for FACS Analysis

Cells flow single file in a stream of fluid Measures multiple characteristics of each cell 10,000 cells are analyzed per reading Flow Cytometry how it works

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. Results: gBK Flow Cytometry Analysis Aripiprazole BupropionSertraline U251 = human glioma Cell IR = Insulin Resistance A = Aripiprazole (10M) B = Bupropion (300M) S = Sertraline (5M)

Results: P11 Flow Cytometry Analysis P11 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 (10M) B = Bupropion (300M) S = Sertraline (5M)

Results: TNF- Flow Cytometry Analysis Glioma insulin resistance (IR) cell line U251 has higher expression of TNF- compared to normal U251 baseline expression. Treatment of Psychotropic Drugs decreases the pro-inflammation cytokine TNF-. U251 = human glioma Cell IR = Insulin Resistance A = Aripiprazole (10M) B = Bupropion (300M) S = Sertraline (5M) TNF-

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 (10M) B = Bupropion (300M) S = Sertraline (5M) IL-6

SDS-PAGE Gel percentage Proteins (-) run towards the anode (+). Separated in pores by size http://www.siumed.edu/~bbartholomew/course_material/protein_methods.htmhttp://www.siumed.edu/~bbartholomew/course_material/protein_methods.htm GE Handbook: Western Blotting Principles and Methods http://bjpsbiotech.edublogs.org/labs-activities/western-blot/ % Gel concentration Pore Size Western Blot

SDS PAGE Transfer to membrane Incubate with antibody http://www.bio.davidson.edu/Courses/genomics/method/Westernblot.html Amrita Ahluwalia, Ph.D. Gastrointestinal(GI) Research Detect antibody Western Blot

Effect of Psychotropic Drugs on gBK protein expression in insulin resistance (IR) glioma cell line U251. Results: Western Blot gBK -Actin 120 kDa

ELISA: Enzyme-Linked Immuno- Sorbent 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

(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.

ELISA: Results

Putting it all together

Pathways by which P11 and gBK mediates behavioral responses to antidepressants protein p11 (also known as S100A10) IL-6 TNF- P11 gBK 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 (10M), Bupropion (300M), and Sertraline (5M) 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 PharmacokineticsBlood and tissue levels Biochemical Modulation of Target 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 ResponseObjective Behavior Response Prolonged Mental Stability Clinical BenefitHappier, better quality of life Phase 1 Phase 2 Phase 3

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University of Bologna Dr. Laura Mercolini Mentor and Professor Laboratory of Pharmaco-Toxicological Analysis; Department of Pharmacy & Biotechnology (FaBiT),