Center for Diabetes & Obesity Research Roseman University of Health Sciences

Research Team: (from right)

Janica C. Wong (Research Associate)

Ronald R. Fiscus (Vice Pres. for Research & Professor of Pharmaceutical Sciences)

Mary G. Johlfs (Director of Research Operations / Scientist)

Ben Costantino (Postdoc Research Assoc.)

New Members (not shown in picture): Oscar B. Goodman, Jr. (Adjunct Professor)

Ranjana Mitra (Research Assist. Professor)

Olivia Chao (Postdoc Research Associate)

Priyatham Gorjala (Postdoc Res. Assoc.)

Supportive Members: Harry Rosenberg (Founding Pres. Emeritus)

Renee Coffman (President & Co-founder)

10 Peer-reviewed Research Articles (2010-2013 ) 3 Book Chapters (2012-2013) 5 Poster Presentations / Abstracts (2010-2013)

Mary Johlfs using advanced nano-proteo-mics (NanoPro 1000) to study pancreatic a- & b-cells, diabetic neuropathies, lung cancer, lymphoma & novel c-Src phosphorylation (pp. 3 & 7)

Type 1 & Type 2 diabetes mellitus (T1D & T2D): Developing better ways of preventing & treating T1D & T2D; Nutra- & pharmaceuticals activating patient’s own stem-cell-recovery. Obesity (How to reverse fat accumulation & prevent T2D). Alzheimer’s disease, ED & other neurological complications of diabetes. Cancers at higher risk in diabetic patients: Blood Cancers (Leukemia, Lymphoma & Myeloma), Breast Cancer, Colon Cancer, Liver Cancer, Lung Cancer, Ovarian Cancer, Pancreatic Cancer & Prostate Cancer. Cardiovascular (CV) diseases associated with T1D & T2D/obesity: Hypertension, Coronary Artery Disease (CAD) & Stroke.

Our research team is dedicated to discovering new (previously unrecognized) molecular mechanisms mediating the pathogenesis of diabetes (both T1D & T2D) & associated pathological complications [peripheral neuropathies, ED, Alzheimer’s, cardiovascular (CV) diseases and diabetes-associated cancers]. Our team is using the most advanced technologies, including TIRF microscopy (p. 5) and ultrasensitive capillary electrophoresis (CE)-based nano-fluidic protein analysis systems [ NanoPro 100 & NanoPro 1000 (nano-proteomics), p. 3, the first of their kind in the Nevada / Utah / Arizona & LA-CA regions of the U.S. ], to find new, more effective ways of preventing & treating diabetes & pathological complications (Alzheimer’s, neuropathies, CV diseases & cancers).

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Pathological Conditions Studied in Our Center:

Our Mission:

Publications during our first 2 ½ Years (details p. 7):

To help support our fight against diseases, contact: rfiscus@roseman.edu or mjohlfs@roseman.edu; Ronald Fiscus (702-427-3139) or Mary Johlfs (702-802-2823), Roseman Reseach Fac.,10530 Discovery Dr, Las Vegas, NV 89135.

[private non-profit health-professional university with 501(c)(3) status]

Las Vegas/Henderson, Nevada

Our Center studies molecular mechanisms mediating pathogenesis of types 1 & 2 diabetes & their pathological complications (see Model above), with special emphasis on developing new therapies for preventing & treating diabetes & the associated pathologies, Alzheimer’s, ED, peripheral neuropathies, CV diseases & cancers.

Awards:

Type 1 & 2 Diabetes Mellitus

Advanced GlycationEnd Products (AGEs)

High glucose levels in blood(Hyperglycemia)

High fat levels in blood(Hyperlipidemia)

Pathological Complications of Diabetes Mellitus

Chronic Inflammation

(Dr. H. Rosenberg,1979 - )

Oxygen free radicals & damage of nitric oxide(NO) / protein kinase G (PKG) signaling pathway

Abnormal lipidutilization & metabolism

Cardiovascular diseases(Hypertension,

Coronary artery disease,Stroke)

Erectile dysfunction(ED)

Neurological complications(Polyneuropathies,

Increased risk ofAlzheimer’s diseases)

Diabetic foot(Foot ulcerations &

amputations)

Diabetic retinopathy& blindness

Increased risk of certain cancers [e.g. breast, colon,

liver, lung, ovarian, pancreatic & prostate cancers

& blood cancers (leukemia, lymphoma & myeloma)]

Roseman University’s new state-of-the-art Research Facility in Summerlin ( far-western/ mountain’s-edge ) district of Las Vegas, Nevada

Janica Wong studying signaling proteins [ CREB, cIAP-1, livin, Mcl-1, PKG-Ia & survinin ] mediat- ing cell survival in bone marrow stem cells, vascular cells & pancreatic a- & b-cells & the exag-gerated cell proliferation & resistance to chemotherapy in lung / ovarian cancer cells (pp. 4 & 7)

Sept. 20, 2012: Dr. Fiscus was awarded the “2012 Nevada Researcher of the Year Award” and Roseman University the “2012 Nevada Research Organization of the Year Award” by the Nevada Biotechnology & Sciences Consortium ( NevBio ).

Nov. 2, 2012: Dr. Fiscus was awarded the “3rd Best Oral Presentation” (out of >100 abstracts) and Mary Johlfs, Janica Wong & Ben Costantino “Best Posters” at The 10th World Congress of Insulin Resistance, Diabetes & Cardiovascular Disease (Los Angeles, CA) (details, pp. 6 & 7).

July 2013: Dr. Fiscus was awarded the “2013 Healthcare Hero / Technol-ogy & Research Award” by Anthem-Blue Cross & Nevada Business mag.

Ben Costantino imaging pancreatic a- & b-cells, cancer cells, neural cells, stem cells & vascular cells using advanced dual-laser TIRF microscopy with live-cell / time-lapse photography (p. 5)

Ronald R. Fiscus, Ph.D., Vice President for Research, Director of the Center for Diabetes &

Obesity Research, Co-Director of Program for Novel Therapeutics in Neurological & Psychiatric Disorders, Head of the Alzheimer’s & Parkinson’s Disease Research Group, Senior Scientist in the Cancer Research Center, Professor of Pharmaceutical Sciences in the College of Pharmacy

Background: • Research training in Analytical Chemistry / Biochemistry at Iowa State University & Molecular Pharmacology at University of California San Diego ( UCSD ) Medical School & Stanford University Medical School, which included the early discovery of nitric oxide ( NO ) & atrial natriuretic peptide ( ANP ) as stimulators of cyclic GMP ( cGMP ) elevation & protein kinase G ( PKG ) activation within intact living cells. Dr. Fiscus’ data provided early evidence of cGMP / PKG signaling pathway as key mediator of NO- & ANP-induced vasodil- ation & antihypertensive effects ( contributing to 1998 Nobel Prize / Medicine, see below).

• Identified cGMP & the PKG-Ia splice variant as key mediators in cell survival & DNA synthe-sis / growth-promoting effects of NO and ANP/BNP at physiological levels [ picomolar ( pM ) to low-nanomolar ( nM ) ], e.g. preventing neural damage caused by toxic levels ( >100 nM )

of NO & reactive oxygen species ( ROSs ) ]. NO ( pM low-nM ) selectively activates PKG-Ia, protecting against neurotoxicity & promoting prolif./chemoresistance in cancer & stem cells.

• Significant contribution to research in Dr. F. Murad’s Lab at Stanford University Medical School, awarded 1998 Nobel Prize in Physiology or Medicine (see publications below).

• Research on early type-5 phosphodiesterase ( PDE5 ) inhibitors ( the prototypes of ViagraTM, CialisTM & LevitraTM ), showing synergistic enhancement of NO’s ability to activate cGMP / PKG signaling pathway & cause subsequent biological effects, now used as highly-successful therapeutic agents for treating erectile dysfunction ( ED ) & pulmonary hypertension.

• 21 years as Professor at Medical Schools, developing new Med. Curriculums & Molecular Biology/Pharmacology Research.

Publications contributing to the 1998 Nobel Prize in Physiology or Medicine ( awarded to Dr. Fiscus’ supervisor at Stanford Medical School, Dr. Ferid Murad, Assoc. Chair, Dept. of Medicine, Stanford Univ., & Chief of Medicine, Palo Alto VA Med. Ctr. ) ( selected from 10 peer-reviewed research articles published with Dr. Murad ) - Establishing cyclic GMP-dependent protein kinase ( protein kinase G, PKG ) as key mediator of NO-, EDRF ( endogenous NO )- & ANP-induced vasodilation / antihypertensive effects.

FISCUS RR, Rapoport RM, Murad F (1983-1984) Endothelium-dependent and nitrovasodilator-induced activation of cyclic GMP-dependent protein kinase in rat aorta. Journal of Cyclic Nucleotide & Protein Phosphorylation Research 9: 415-425. FISCUS RR, Rapoport RM, Waldman SA, Murad F (1985) Atriopeptin II elevates cyclic GMP, activates cyclic GMP-dependent protein kinase and causes relaxation in rat thoracic aorta. Biochimica et Biophysica Acta 846: 179-184. Murad F, Rapoport RM, FISCUS RR (1985) Role of cyclic GMP in relaxations of vascular smooth muscle. Journal of Cardiovascular Pharmacol- ogy 7 Suppl 3:S111-S118. FISCUS RR, Robles BT, Waldman SA, Murad F (1987) Atrial natriuretic factors stimulate accumulation and efflux of cyclic GMP in C6-2B rat glioma and PC12 rat pheochromocytoma cell cultures. Journal of Neurochemistry 48: 522-528. FISCUS RR, Murad F (1988) cGMP-dependent protein kinase activation in intact tissues. Methods in Enzymology 159: 150-159. FISCUS RR, Zhou HL, Wang X, Han C, Ali S, Joyce CD, Murad F (1991) Calcitonin gene-related peptide (CGRP)-induced cyclic AMP, cyclic GMP and vasorelaxant responses in rat thoracic aorta are antagonized by blockers of endothelium-derived relaxant factor (EDRF). Neuropeptides 20: 133-143.

Mary G. Johlfs, M.S, Director of Research Operations / Scientist

Background:

Mary Johlfs received her Masters degree in Biological Sciences from Northern Illinois University before starting a research position in the biotechnology industry at PDL Biopharma. Mary Johlfs has also held a research and managerial position at the University of Illinois at Chicago and the Nevada Cancer Institute ( NVCI ), Las Vegas, NV. At the NVCI, Mary Johlfs was the Lab Manager and Senior Research Associate in Dr. Fiscus’ lab , while Dr. Fiscus served as Director of the Cancer Molecular Biology Section at NVCI from mid-2006 to the end of 2009. Mary Johlfs has over 14 years of research experience in the fields of cellular and molecu-lar biology. She also has vast experience with state-of-the-art research instrumentation (e.g. NanoPro 100 and NanoPro 1000 systems for ultrasensitive quantification of protein expression and protein phosphorylation, details on p. 3). Mary serves as the Director of Research Operations for all Roseman Research Centers & Programs and is a top-ranked / extensively-published Scientist at Roseman University of Health Sciences (pp. 3 & 7).

Management Team:

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PKG-Iαand PKG-Iβexpression

in NCI-H2052 mesothelioma

cells detected by Western blot &

the NanoPro100 system

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Recombinant PKG-Iαand PKG-Iβ

detected with the

NanoPro100 system

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5.0 5.5 6.0 6.5

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PKG-Iαprotein detected in a

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Mary Johlfs operating the NanoPro 1000 system ( advanced nano-proteomics ), a capillary-electrophoresis ( CE )-based nano-fluidic immuno-detection system for ultrasensitive ( low-femtogram-level ) measurements of protein expression & phosphorylation levels in biological samples ( >500-times the sensitivity of Western blots , used by other labs around the world ).

Our new NanoPro 1000 system and smaller Nano-Pro 100 system (set up at Roseman 2 years ago) represent the first instruments of their kind in the Nevada/Utah/Arizona & LA-CA regions of the U.S.

Illustration of the exquisite sensitivity & separation capabilities of the NanoPro 100 system, able to quantify proteins with a sensitivity >100 times better than conventional Western blot analysis (used by other labs). Our newer, more advanced NanoPro 1000 system has an additional 5 times higher sensitivity (i.e. >500 times better than conventional Western blot analysis), thus allowing discovery of previously unrecognized proteins in cells.

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Purified PKG-Ia

NG108-15 lysate

anti-PKG-Ia/β Ab

A. PKG-Ia/b protein expression analysis by traditional Western blot

PKG-Ia/b expression analysis of NG108-15 cells using NanoPro100 system (Expanded view of the X-axis

to show shift in pI values)

B.

PKG-Ia

pI

PKG-Ia

pI

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C. PKG-Ia/b expression analysis by NanoPro100 system of the NG108-15cell lysate treated with lambda phosphatase

(Expanded view of the X-axis to show shift in pI values)

Figure 8

Neuronal cell line NG108-15 expresses exclusively the PKG-Ia splice variant of PKG-I, determined by NanoPro 100 system. This instrument also detects phosphorylated forms of PKG-Ia . Data from our book chapter in “Protein Kinase Technology” in NEUROMETHODS (Fiscus & Johlfs, 2012) (details p. 7). PKG-Ia is essential for protecting the survival & functions of neural cells.

Johlfs, M.G. and R.R. Fiscus. (2010) Protein kinase G type-Ia phosphorylates the apoptosis-regulating protein BAD at serine 155 and protects against apoptosis in N1E-115 cells. Neurochemistry International 56: 546-553.

Fiscus RR & Johlfs MG (2012) Protein kinase G (PKG): Involvement in promoting neural cell survival, proliferation, synaptogenesis, and synaptic plasticity and the use of new ultrasensitive capillary-electro-phoresis-based methodologies for measuring PKG expression and molecular actions. Chapter 18, in “Protein Kinase Technologies” (ed. H. Mukai) in NEUROMETHODS, Vol. 68, Springer, pp. 319-348..

Fiscus, R.R. (2002) Involvement of cyclic GMP and protein kinase G in the regulation of apoptosis and survival of neural cells. Neurosignals 11: 175-190.

Our Center studies the neurological complications of diabetes & obesity, including Alzheimer’s diseases & peripheral nerve damage caused by high-fat / high-glucose levels and insulin resistance. Previously, our lab identified the Ia isoform of PKG ( PKG-Ia ) as a key mediator of cell survival & neuroprotection, preventing cellular damage caused by high / toxic levels ( >100 nM ) of NO, oxygen free radicals & other reactive oxygen species (ROSs).

At Roseman University, we use state-of-the-art instrumentation [e.g. NanoPro 100 & 1000 systems (this page) and TIRF micro-scopy (details, p. 5)] as ultrasensitive technologies for determining the role of previously-unrecognized proteins in the cyto-protection of neurons / b-cells / stem cells against toxicity of high-level NO & ROSs, preventing pathogenesis of diabetes, neuropathies & CV dis- eases, & the exaggerated proliferation & chemoresistance in cancers.

Discovery of novel, previously unrecognized proteins involved in neuroprotection & improved survival of pancreatic a- & b-cells ( preventing Alzheimer’s disease, Parkinson’s disease, diabetic neuropathies & damage of a- & b-cells during diabetes ) and in abnormal proliferation & chemoresistance in cancer cells.

cGMP

Decreased release ofcytochrome C

INHIBITION OF APOPTOSISINHIBITION OF APOPTOSIS

NO

(physiological levels: 0.5 – 10 nM)

Growth/Survivalfactors

Decreased activity of caspase-3

Growth/Survivalfactors

INCREASED PROLIFERATIONINCREASED PROLIFERATION

sGC

heme PKG-Ia

CREBser133P

Increased expression of anti-apoptotic

proteins

NucleusNucleusNucleus

eNOS

PDK1PI3KAkt

CytosolCytosolCytosol

P

GFR

P

GFRser1177HSP90

SH3

Caveolin-1

NO

Model of N0/PKG-Ia-mediated stimulation of cancer cell survival and proliferation

Endothelial cell

Pser17

P

SrcRaf-1

MEK1/2

ERK1/2

Model prepared by Mary G. Johlfs,Dr. R.R. Fiscus’ Lab, Nevada Cancer Inst.

PI3K

Akt

eNOS

PDK1

ser1177P

ser1177

PKG-Ia

PKG-Ia

SH2

CytosolicSequestration

P

BAD

Bcl-2

Raf-1

Bcl-XL

ser112ser136

P

ser112P

14-3-3

BAD

ser155P

Mitochondrion

HSP90

Caveolin-1

VEGFR2/Flk1

ser136

P

P

ser155

Ras

VEGF

Cancer cell of epithelial origin

(expressing eNOS), e.g. breast, lung,ovarian and prostate cancer

sGC

heme

NO

cGMP

Cell survival

Angiogenesis

Ras

FGFR bFGF

MEK1/2

ERK1/2

Raf-1

PKG-Ia

Proliferation, Migration, Tube formation

PKG-Ia

Mitochondrion

PKA

cAMP

Angiopoietin-1

SDF-1a

(Enhanced Tumor Growth & Chemoresistance)

Angiogenic Switch

PTEN

Cellular model illustrating our data identifying the role of nitric oxide (NO) and downstream activation of PKG-Ia in cancer cells of epithelial origin (including breast, lung, ovarian and prostate cancers as well as mesothel-ioma). The NO / cGMP / PKG-Ia signaling pathway plays a central role in promoting c-Src activation & the exaggerated DNA synthesis / cell prolifer-ation of cancer cells as well as resistance to chemotherapeutic agents like cisplatin (chemoresistance) in certain resistant populations of cancer cells. The exaggerated proliferation & chemoresistance can result in a relapse of tumor growth following chemotherapy. Also illustrated above is the key role of the NO / cGMP / PKG-Ia signaling pathway in vascular endothelial cells, promoting angiogenesis (new blood vessel growth).

Janica Wong is determining the role of the NO / cGMP / PKG and ANP / cGMP / PKG signaling pathways in regulating DNA synthesis / cell proliferation, cell migration and resistance to apoptosis (promoting cell survival) in both normal non-cancerous cells [ e.g. OP9 bone marrow-dervied mesenchymal (stromal) stem cells, vascular endothelial cells / smooth muscle cells and pancreatic a- & b-cells ] and cancer cells [ ovarian cancer and non-small cell lung cancer (NSCLC) cells ].

Janica’s data has shown that NO at low physiological levels (0.01 – 10 nM NO) promotes DNA synthesis / cell proliferation and cell survival in vascular smooth muscle cells (VSMCs) via a mechanism involving stimulation of PKG-Ia kinase activity. Higher levels of NO, which additionally activate the other splice variant of PKG-I ( i.e. PKG-Ib ) and other NO-mediated mechanisms (e.g. S-nitrosylation and tyrosine-nitration of proteins), have the opposite effect, inhibiting cell proliferation & inducing apoptotic cell death. Interestingly, prior activation of the cytoprotective PKG-Ia by natriuretic peptides (ANP / BNP) dramatically protects VSMCs against toxic / pro-apoptotic effects of high-level ( >100 nM ) NO (Wong & Fiscus, 2010).

Wong, J.C. and R.R. Fiscus. (2010) Protein kinase G activity prevents pathological-level nitric oxide-induced apoptosis and promotes DNA synthesis / cell proliferation in vascular smooth muscle cells. Cardiovascular Pathology 19: e221-e231. (Online)

Janica has also found a key role of low-level NO & downstream PKG-Ia activation in promoting DNA synthesis / cell proliferation and protecting against apoptosis in OP9 bone marrow-derived mesenchymal (stromal) stem cells, human lung & ovarian cancer cells and mouse pancreatic b-cells. The role of PKG-Ia was defined by using both pharmacological inhibition of kinase activity and genetic modulation using gene knockdown with siRNA targeting PKG-Ia.

Leung, E.L., Wong, J.C., Johlfs, M.G., Tsang, B.K., and R. R. Fiscus. (2010) Protein kinase G type Ia activity in human ovarian cancer cells significant-ly contributes to enhanced Src activation and DNA synthesis/cell proliferation. Molecular Cancer Research 8 (4): 578-591.

Wong, J.C. and R.R. Fiscus. (2011) Essential roles of nitric oxide (NO)/cGMP/protein kinase G type-Ia (PKG-Ia) signaling pathway and atrial natriuretic peptide (ANP)/cGMP/PKG-Ia autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells. Journal of Cellular Biochemistry 112: 829-839.

Wong, J.C., Bathina, M., and R.R. Fiscus. (2012) Cyclic GMP/protein kinase G type-Ia (PKG-Ia) signaling pathway promotes CREB phosphoryl-ation and maintains higher c-IAP1, livin, survivin and Mcl-1 expression and the inhibition of PKG-Ia kinase activity synergizes with cisplatin in non-small cell lung cancer cells. Journal of Cellular Biochemistry 113: 3587-3598.

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Picomolar-to-low-nanomolar-NO / cGMP / PKG-Ia signaling pathway also plays a key role in promoting DNA-synthesis / cell proliferation, cell migration & cell survival (i.e. chemoresistance) in vascular cells, bone marrow-derived stem cells and many cancer cells.

NCI-H23 cells [non-small cell lung cancer (NSCLC) cells]. NSCLC is the most common form of lung cancer.

TIRF microscopy image of NCI-H23 lung cancer cells, with fluorescent staining for PKG-Ia (green).

The above image shows subcellular localization of PKG-Ia at the plasma membrane (as illustrated in the cellular model on page 4). Our current experiments are determining the co-localization of PKG-Ia with other oncogenic proteins, e.g. c-Src (a non-receptor tyrosine kinase) and mutated EGFR, common targets of newly-developed anti-cancer therapies.

Ben Costantino operating Roseman’s TIRF (total internal reflection fluorescence) microscope, possessing a unique laser system that generates an evanescent wave of light that illuminates only the closest 100 nanometers (i.e. 0.1 microns ), only at the cell’s plasma membrane, thus improving the signal-to-noise-ratio by 100 fold. This allows imaging of proteins and other signaling molecules near the cell membrane with a sensitivity much better than conventional microcopy or confocal microscopy.

Bone marrow-derived mesenchymal (stromal) stem cells (BM-MSCs) , multipotent cells capable of differ-entiating into fat cells, bone cells or cartilage cells, used at Roseman University as a model of obesity & type-2 diabetes (with differentiation of BM-MSCs into fat cells). Shown above are TIRF microscope images of these cells, featured on the cover of the Spring 2012 issue of Roseman’s Remedy magazine.

Undifferentiated BM-MSC ( OP9 cell ), used as a “pre-adipocyte”. Green staining represents PKG-Ia localized at the plasma membrane (by TIRF microscopy) and blue staining represents the nucleus (by wide-field fluorescent microscopy). Merged image.

These BM-MSCs are partially differentiated into fat cells. Note that some cells contain lipid droplets, whereas others do not. Interestingly, PKG-Ia is pushed out of the way by the growing lipid droplets.

Differentiated adipocyte ( fully-developed fat cell ) that has originated from a bone marrow-derived mesenchymal stem cell (our cellular model used for developing new therapies for preventing & treating obesity / Type-2 diabetes).

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Ben is also developing a novel technique for quantifying protein kinase activity in biological samples & recombinant proteins, using a new near-infrared fluorescence (NIRF)-based methodology, rather than the older technique requir-ing use of radioactive material. Our new NIRF-kinase-assay methodology provides sensitivity and accuracy similar to the older 32P-based assays *the ‘gold standard’ of kinase assays, used by Dr. Fiscus for quantifying PKA and PKG kinase activity while a postdoc / scientist at UCSD and Stanford University Schools of Medicine (see publications listed on p. 2)]. Our new methodology has the unique feature of being able to quantify the intracellular kinase activities of both PKG & protein kinase C (PKC) simultaneously & selectively.

Ferid Murad, MD, PhD (1998 Nobel Prize in Physiology or Medicine), Professor of Biochemistry & Molecular Biology, George Washington University, Washington, D.C. & Director, Murad Research Institute, Shanghai, China. (Former supervisor of Dr. Fiscus at Stanford University Medical School). Collaborative project: “PKG Isoforms in Glioma Cells”.

Chikao Morimoto, MD, PhD, Professor & Chair, Department of Clinical Immunology, & Director, Advanced Clinical Research Center, University of Tokyo, Tokyo, Japan, & Visiting Professor, College of Pharmacy, Roseman University of Health Sciences. Dr. Morimoto is a world expert on CD26 / dipeptidyl peptidase type-IV (DPP-IV) protein and its involvement in immunology, diabetes & cancer. Collab. project: “Molecular Mechanism of CD26 / DPP-IV-Targeted Therapies in Cancer & Diabetes”.

Maria Grant, MD, Professor, Dept. of Pharmacology & Therapeutics, College of Medicine, University of Florida, Gainesville, FL. Dr. Grant is a leader in Diabetic Retinopathy, with special interest in the role of NO and its stimulation of the phosphorylation of VASP (a PKG substrate & regulator of actin filament / focal adhesion) in endothelial cells and endothelial progenitor cells (EPCs).

Dr. Fiscus serves on the Scientific Advisory Board for the NIH Program Project Grant (NIH P01 grant) on “Diabetic Retinopathy” at the University of Florida, headed by Dr. Grant. Collaborative project: “NanoPro analysis of cytoprotective PKG isoforms in EPCs”.

Elaine L. Leung, PhD, Assistant Professor, State Key Laboratory of Chinese Medicines, Macau University of Science & Technology (MUST), Macau Special Admin. Region, China. Dr. Leung is a former Ph.D. student & Postdoc supervised by Dr. Fiscus. Collab. projects: “Cancer Stem Cells (CSCs) as the ‘Seeds’ of Lung Cancer” & “New Treatments for Diabetes: Improved Survival of Isolated Pancreatic b-cells for Transplantation Therapy”.

Maria P. Wong, MD, Associate Professor, Department of Pathology, The University of Hong Kong, Faculty of Medicine, Hong Kong Special Admin. Region, China. Collaborative project: “Identifying Cancer Stem Cells as the Origin of Lung Cancer in Patients”.

Thuc (Tim) Le, PhD, Assistant Professor, Nevada Cancer Institute (NVCI)/Desert Research Institute (DRI), Las Vegas, NV. Dr. Le is a leading scientist in the use of 4-photon/CARS microscopy for lipid imaging and determination of lipid metabolism. Recent publication, based on our collaborative work: Urasaki, Y., Johlfs, M.G., Fiscus, R.R. and T.T. Le. (2012) Imaging immune and metabolic cells of visceral adipose tissues with multimodal nonlinear optical microscopy. PLoS ONE (June 2012) 7(6): e38418.

Oscar B. Goodman, Jr., MD, PhD, Comprehensive Cancer Centers of Nevada, Las Vegas, NV, & Adjunct Professor, Roseman University of Health Sciences. Dr. Goodman is a leading clinician/scientist studying the isolation & characterization of circulating tumor

cells (CTCs). Collab. Project.: “NanoPro Analysis of CTCs – Ultrasensitive/Quantitative Tool for Personalized Medicine”.

Aurelio Lorico, MD, PhD & Germana Rappa, MD, PhD, Co-Directors of Roseman’s Cancer Research Program, Associate Professors, College of Pharmacy, Roseman University of Health Sciences, Henderson/Las Vegas, NV. Dr. Lorico & Dr. Rappa are leaders in the field of CD133 protein in stem cell biology and the interdependence between cancer cells and bone marrow-derived stem cells. Collab. Project: “NanoPro Analysis of CD133 & b-catenin in Cancer & Stem Cells”.

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Invited Oral Presentations Given by Dr. Fiscus (Representing Roseman U. Around the World): Nov. 29, 2010 . Novel role of nitric oxide / cyclic GMP / protein kinase G type-Iα (PKG-Iα) signaling pathway in promoting cell survival and DNA synthesis / proliferation in various types of cancer cells and stem / progenitor cells. Presented to the NIH Program Project Grant (NIH PO1) group studying molecular mechanisms of diabetic retinopathy, directed by Dr. Maria Grant, M.D., Professor, Department of Pharmacology & Therapeutics, College of Medicine, University of Florida, Gainesville, FL.

June 26, 2011. Protein kinase G type-I phosphorylates c-Src at serine-17 and promotes cell survival, proliferation and attachment in human mesothelioma and non-small cell lung cancer cells. 5th International Conference on Cyclic GMP (cGMP): Generators, Effectors and Therapeutic Implications, Halle, Germany, June 24-26, 2011.

Sept. 26, 2011. PKG-Iα activation by picomolar-to-low-nanomolar NO or ANP promotes DNA synthesis / cell proliferation & protects neural and vascular cells against the toxic / pro-apoptotic effects of high-level NO (model of cytoprotection during inflammation). 2nd International Conference of Inflammation & Translational Medicine, Shanghai, China, Sept. 25-26, 2011.

Oct. 7, 2011. Novel role of the nitric oxide / cGMP / PKG-Iα signaling pathway in promoting cell survival and proliferation in neural, stem / progenitor and cancer cells: Quantification using the NanoPro system. NanoPro User’s Meeting, Washington, D.C., Oct. 5-7, 2011.

March 12, 2012. Keynote Address - Discovery of the nitric oxide (NO) / cGMP / protein kinase G (PKG) signaling pathway: Established role in cardiovascular diseases and erectile dysfunction and emerging role in cancer and stem cell biology. Touro University-Nevada Annual Research Day, Henderson, NV.

Nov. 2, 2012. Involvement of physiological-level nitric oxide (NO)/protein kinase G type-Ia (PKG-Ia) signaling in protecting against diabetic neuropathies: Glucose effects on PKG-Ia expression and downstream signaling quantified by ultrasensitive capillary-electrophoresis (CE)-based NanoPro 1000 protein analysis system. 10th World Congress of Insulin Resistance,

Diabetes & Cardiovascular Disease, Los Angeles, CA, Nov. 1-3, 2012. (Awarded 3rd Best out of over 100 abstracts.)

Collaborations with 9 Top-rated Scientists from Around the World:

To help support our fight against diseases, contact: rfiscus@roseman.edu or mjohlfs@roseman.edu; Ronald Fiscus (702-427-3139) or Mary Johlfs (702-802-2823), Roseman University, Research Facility, 10530 Discovery Drive,

Las Vegas, NV 89135. Roseman University is a private non-profit health-professional-degree-granting university with 501(c)(3) status.

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Johlfs, M.G. and R.R. Fiscus. (2010) Protein kinase G type-Ia phosphorylates the apoptosis-regulating protein BAD at serine 155 and protects against apoptosis in N1E-115 cells. Neurochemistry International 56: 546-553.

Wong, J.C. and R.R. Fiscus. (2010) Protein kinase G activity prevents pathological-level nitric oxide-induced apoptosis and promotes DNA synthesis / cell proliferation in vascular smooth muscle cells. Cardiovascular Pathology 19: e221-e231. (Online Publishing)

Leung, E.L., Wong, J.C., Johlfs, M.G., Tsang, B.K., and R. R. Fiscus. (2010) Protein kinase G type Ia activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis / cell proliferation. Molecular Cancer Research 8 (4): 578-591.

Leung, E.L, Fiscus, R.R., Tung, J.T., Tin, V.P., Cheng, L.C., Sihoe, A.D., Fink, L.M., Ma, Y. and M.P. Wong. (2010) Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS ONE 5 (11): e14062. (Online Publishing)

Wong, J.C. and R.R. Fiscus. (2011) Essential roles of nitric oxide (NO) / cGMP / protein kinase G type-Ia (PKG-Ia) signaling pathway and atrial natriuretic peptide (ANP) / cGMP / PKG-Ia autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells. Journal of Cellular Biochemistry 112: 829-839.

Mitra, R., Chao, O.S., Nanus, D.M. and O.B. Goodman, Jr. (2012) Negative regulation of NEP expression by hypoxia. Prostate DOI 10.1002/pros.22613 (pages 1 – 9).

Mitra, R., Chao, O., Urasaki, Y., Goodman, O.B., and T.T. Le (2012) Detection of lipid-rich prostate circulating tumour cells with coherent anti-Stokes Raman scattering microscopy. BMC Cancer 12: 540 (pages 1 – 9).

Urasaki, Y., Johlfs, M.G., Fiscus, R.R. and T.T. Le. (2012) Imaging immune and metabolic cells of visceral adipose tissues with multimodal nonlinear optical microscopy. PLoS ONE (June 2012) 7 (6): e38418. (Online Publishing)

Wong, J.C., Bathina, M., and R.R. Fiscus. (2012) Cyclic GMP / protein kinase G type-Ia (PKG-Ia) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin and Mcl-1 expression and the inhibition of PKG-Ia kinase activity synergizes with cisplatin in non-small cell lung cancer cells. Journal of Cellular Biochemistry 113: 3587-3598.

Rappa, G., Mercapide, J., Anzanello, F., Le, T.T., Johlfs, M.G., Fiscus, R.R., Wilsch-Brauninger, M., Corbeil, D. and A. Lorico. (2013) Wnt interaction and extracellular release of prominin-1 / CD133 in human malignant melanoma cells. Experimental Cell Research 319: 810-819.

Research Articles (peer-reviewed):

Book Chapters: Fiscus, R.R., Leung, E.L., Wong, J.C. and M.G. Johlfs. (2012) Nitric oxide / protein kinase G-Ia promotes c-Src activation, proliferation and chemoresistance in ovarian cancer. Chapter 16 in “Ovarian Cancer – Basic Science Perspective”, ed. S. Farghaly, INTECH Open Access Publishing, pp. 315-334.

Fiscus, R.R. and M.G. Johlfs. (2012) Protein kinase G (PKG): Involvement in promoting neural cell survival, proliferation, synaptogenesis and synaptic plasticity and the use of new ultrasensitive capillary-electrophoresis-based methodology for measuring PKG expression and molecular actions. Chapter 18 in “Protein Kinase Technologies”, ed. H. Mukai, in the series NEUROMETHODS, Vol. 68, Springer, pp. 319-348.

Wong, J.C. and R.R. Fiscus. (2013) Protein kinase G-Ia hyperactivation and VASP phosphorylation in promoting ovarian cancer cell migration and platinum resistance. Chapter 12 in “Ovarian Cancer – A Clinical and Translational Update”, ed. I. Diaz-Padilla, INTECH Open Access Publishing, Section 4. Molecular Pathogenesis and Targeted Therapies, pp. 251-273. . Poster Presentations (last two presented):

Johlfs, M.G., Coffman, R., Rosenberg, H. and R.R. Fiscus. (2012) Involvement of physiological-level nitric oxide (NO)/protein kinase G type-Ia (PKG-Ia) signaling in protecting against diabetic neuropathies: Glucose effects on PKG-Ia expression and downstream signaling quantified by ultrasensitive capillary-electrophoresis (CE)-based NanoPro 1000 protein analysis system. 10th World Congress of Insulin Resistance, Diabetes & Cardiovascular Disease, Los Angeles, CA, Nov. 1-3, 2012. (Special Award–Top Poster Presentations out of >100)

Wong, J.C., Coffman, R., Rosenberg, H. and R.R. Fiscus. (2012) Essential role of the nitric oxide (NO) / protein kinase G type-Ia (PKG-Ia) signaling pathway in promoting the survival and insulin secretion of pancreatic b-cells. 10th World Congress of Insulin Resistance, Diabetes & Cardiovascular Disease, Los Angeles, CA, Nov. 1-3, 2012. (Special Award–Top Poster Presentations out of >100)

Publications & Poster Presentations from Our Center During Its First 2 ½ Years