Biographical Sketch

Education,   Advanced  Medical  Research  Training  at  UCSD  &  Stanford  University  Medical  Schools,  &  Highlights of Accomplishments / Awards (chronological):

• B.S. ( Chemistry major,  Math & Physics Minors),  special  training  in  Analytical  Chemistry / Quantitative  Analysis  by  Dr. Harvey Diehl [Dept. Chemistry, Iowa State University of Science & Technology (ISU-S&T)]) &  overall  Supervision  by  Dr. Velmer A. Fassel  (Dept. Chemistry, ISU-S&T &  Ames  Laboratory - a U.S Department of Energy (DOE)  National  Lab ,  plus  2 years  of  course-work in German language (spoken & written language)  &  special  training  in  translation  of  German-language  technical / chemistry  documents,  Iowa State University of Science & Technology (ISU-S&T),  Ames,  IA.

• M.S. ( Neurophysiology, specialty in Neurochemistry ),  Thesis:   Quantification ( using  ultra-sensitive  fluorescence  technology )  of  Dopamine  (DA)  &  Norepinephrine  (NE)  turnover in brain regions ( e.g.  hippocampus, cerebral cortex, corpus striatum, hypothalamus, brain stem ) in animal model of acetylcholine esterase  ( AChE ) inhibitor exposure  ( PARATHION )  &  subsequent  activation  of  cholinergic  neural  activity  in  the  brain (Model of Alzheimer's & Parkinson's neuropathology),  Supervisor:  Dr. William Van Meter,  Iowa State University of Science & Technology, Ames, IA. 

• Ph.D. ( Molecular Pharmacology/Physiology,  minor: Biochemistry ),   Dissertation:    Nitroglycerin,  a  Nitric Oxide ( NO ) mimetic,  stimulates  cyclic GMP ( cGMP )  elevations  and  Prostaglandin E1 & E2  ( PGE1  &  PGE2 )  stimulate  cyclic AMP ( cAMP )  elevations,  mediating  the vasodilation / contractile changes  in  human  isolated  arteries (including human umbilical arteries).  Supervision:  Dr. Donald C. Dyer,  Iowa State University of Science & Technology, Ames, IA.

• Advanced research training  as a Postdoctoral Fellow (Molecular Pharmacology) in the lab of  Dr. Steven E. Mayer ( President of the American Society of Pharmacology & Experimental Therapeutics (ASPET) & one of the founding fathers of the field of Molecular Pharmacology),  Founding Chair, Division of Pharmacology, Department of Medicine,  University of California San Diego ( UCSD ) School of Medicine, La Jolla, CA.    My final year at UCSD Medical School was SUPERVISED by Dr.  Palmer Taylor, former Chair of Pharmacology, UCSD Medical School, & founding Dean of the newly-established School of Pharmacy, UCSD.

• Competed for & was successfully awarded 4 consecutive 1-year Individual/Competitive Postdoctoral Fellowships, 2 from American Heart Assoc. (AHA) & 2 from Muscular Dystrophy Assoc. (MDA), supporting my Postdoc research training with Dr. Steven E. Mayer at UCSD Medical School & my first year with Dr. Ferid Murad, Assoc. Chair, Department of Medicine, Stanford University Medical School & Chief of Medicine, Palo Alto Veterans Admin. (VA) Medical Center, Palo Alto, CA.  

• Fellow in Clinical Pharmacology, Stanford University School of Medicine  &  Scientist,  Palo Alto Veterans Admin. (VA) Medical Center,  in lab of Dr. Ferid Murad (Nobel Laureate),  Associate Chair (& later Acting Chair) of  Dept.  of  Medicine,  Stanford  University  Medical  School,  &  the Chief of Medicine, Palo Alto VA Medical Center, Palo Alto, CA.

Contributed to research projects involved in the awarding of the 1998 NOBEL PRIZE in Physiology or Medicine to my supervisor, Dr. Ferid Murad at Stanford  University Medical School / Palo Alto VA Medical Center.  I served as  LEAD SCIENTIST  in pioneering the development of novel methodologies for protein kinase analysis ( PKA & PKG ), resulting in:         1)  identifying  Protein Kinase G ( PKG ) as the key downstream target and mediator of vascular responses to  healthy-level  Nitric Oxide ( NO ),  called  Endothelium-Derived Relaxant Factor ( EDRF ) at the time,  and responses to the cardiac hormone,  atrial  natriuretic  peptide ( ANP )  (see Fiscus et al. 1983, Fiscus et al. 1985,  &  8  other publications with Dr. Murad),         2)  showing that acetylcholine ( ACh )-triggered release of EDRF ( now recognized as endogenous, healthy-level NO ) from vascular endothelial cells (generated by eNOS) can  selectively  increase  the  PKG  kinase/catalytic  activity  in  nearby  cells [ in this tissue model, vascular smooth muscle cells ( VSMCs )], causing vasodilation & protection of the cardiovascular system, now recognized to prevent many cardiovascular diseases (Fiscus et al., 1983; Fiscus & Murad, 1988;  Fiscus, 1988),        3)  showing that basal-level release of NO (in absence of eNOS stimulator) from vascular endothelial cells is sufficient to significantly elevate the catalytic/kinase activity of PKG in nearby cells  (basal levels of NO are now measured to be 0.01 - 1 nM, sufficient to partially activate PKG & provide cell-survival & regenerative capabilities in mammalian cells),        4)  showing that physiological-levels  of both  NO  &  ANP,  when added to both  neuronal cells  &  glial cells,  stimulate cyclic GMP (cGMP) biosynthesis, elevating cGMP levels in both intracellular & extracellular compartments of neuronal & glial cells  (this evidence helped to establish the role of healthy-level NO & ANP as key regulators of both neuronal- and glial-type neural cells via the ability to elevate intracellular cGMP and then activate downstream PKG kinase activity (Fiscus et al., 1987), now recognized as essential for healthy function, survival & regeneration / repair of neural cells (Fiscus & Johlfs, 2012, book chapter in Protein Kinase Technology in Neuromethods), & the concept that extracellular cGMP elevations , stimulated by NO or Natriuretic Peptides (ANP, BNP, CNP) , can serve as a cell-to-cell communication signal regulating neuronal functions,          5)  developing the research ideas,  designing the research experiments,  conducting the lab research & analyzing the data in the Murad Lab, Stanford Univ.  and      6)   publishing 10 peer-reviewed scientific articles with Dr. Murad (1983 - 1991).          

This 1998 Nobel Prize involved  “Discovery of NO physiology, pharmacology and pathobiology & the involvement of its signaling pathway, i.e. healthy-level / cardiovascular-protective form of  NO ( via elevation of cGMP levels and activation of PKG catalytic/kinase activity) and the specific protective role of vascular endothelium-derived NO, via its selective activation of PKG, to protect against various cardiovascular diseases (e.g. hypertension, atherosclerosis, coronary artery disease) ".   This signaling pathway, involving cGMP / PKG and  its  anti-inflammatory  &  cardiovascular- / multiple organ system- protective effects,  is now recognized to be damaged by the pathology of diabetes (both type-1 and type-2 diabetes, T1D & T2D ) and by other causes of chronic inflammation (as occurs when there is high levels of fats in the diet, i.e. HFD,  even without the clear signs of diabetes )  ( see our 2017 publications in Diabetes & Vascular Disease Research showing essential role of the PKG1alpha splice variant in promoting cell survival & regeneration of Pancreatic Beta-cells via downstream phosphorylation & regulation of Akt & FoxO1 ).   Damage of cGMP / PKG  causes  loss of its  cyto/neuro-protective  and  anti-inflammatory actions,  thus contributing to the pathogenesis of many pathologies,  best recognized in cardiovascular diseases (CVDs) and erectile dysfunction ( ED ), but also neurological damage, e.g. diabetic peripheral neuropathies (DPNs) & delayed recovery from traumatic brain injury (TBI), chronic traumatic encephalopathy (CTER), & spinal cord injury (SCI) & the development of PTSD, Migraine-like headaches, depression. addictions & later Alzheimer's-like  dementia.

• 34 Research Grants successfully funded over a 21-year career as Assistant, Associate & Full Professor at Medical Schools.

• Published 88 peer-reviewed scientific articles, 8 book chapters, & gave over 100 scientific presentations.

• Mentored/Supervised the research training of:   8 Postdoctoral Fellows,  7 PhD graduate students,  4 MS graduate students, 4 medical students, 2 undergraduate students & 2 visiting scientists over the last 35 years, while conducting medical research at  UCSD School of Medicine,  Stanford University School of Medicine,  Loyola University of Chicago Stritch School of Medicine,  University of Kentucky College of Medicine,  Chinese University of Hong Kong (CUHK) Faculty of Medicine,  Nevada Cancer Institute (NVCI)  &  Roseman University of Health Sciences Colleges of Pharmacy & Medicine.  

• Inventor on 2 patent applications ( one Awarded on 3-17-2020 ) – Developing advanced proteomic technologies (exquisitely-sensitive/more-versatile proteomics) for the discovery of novel biomarkers of various diseases, e.g. Alzheimer's disease (AD), cardiovascular diseases (CVDs), erectile dysfunction (ED) & type-1/type-2 diabetes [based on splice variant switches & post-translational modifications (phosphorylations, acetylations, glycosylation, nitrations)] & the development of new, better therapeutic agents for effective/specific molecular targeting in the prevention & treatment of these diseases (i.e. new way for analyzing protein biomarkers of various pathologies & developing new therapies based on personalized/targeted therapeutics).  

• 21 years as faculty member at medical schools, teaching medical students & establishing Research Programs on Molecular/Translational Pharmacology, Molecular Pathology & Analytical Chemistry/Biochemistry of Cardiovascular diseases, Type-1/type-2 Diabetes, Cancers & Neurological pathologies, including Traumatic Brain Injury (TBI) and Stroke, and subsequent development of Post-traumatic Stress Disorder (PTSD), Depression, Addictions and, later, Alzheimer's Disease (AD).

• Awarded the “2012  Researcher of the Year Award”  for Southern Nevada, given by the Nevada Biotechnology & Sciences Consortium (Nev-Bio), shared with two other scientists in Southern Nevada, Dr. Martin Schiller, Director of the Nevada Institute of Personalized Medicine, University of Nevada Las Vegas (UNLV), & Dr. Robert Webber, Founder & CEO of R&D Antibodies, Las Vegas.

• Awarded the “2013  HealthCare Hero–Research & Technology Award” for Southern Nevada, given by the Nevada Business Magazine & Blue Cross/Blue Shield of Nevada.

• 2006 - 2010, Director of Cancer Molecular Biology section, Nevada Cancer Institute (NVCI), Summerlin-Las Vegas, NV.

• 2006 - 2019, Adjunct Professor, Dept of Physiology & Pharmacology, Perinatal Biology Inst., Loma Linda University Medical School.

• 2010 - 2015,  First University-wide Director of Research & first Vice President for Research (VPR) at Roseman University's three campuses:  Henderson, NV-Campus, Summerlin-Las Vegas, NV-Campus & South Jordan, UT-Campus) & the Director of Research for Diabetes & Obesity Research Program, Roseman University of Health Sciences (Headquarters in Henderson, NV). 

• 2010 - 2018,  Professor of Pharmaceutical Sciences, College of Pharmacy, Roseman University.

• 2015 - 2017, First Associate Dean of Research, College of Medicine, Roseman University.

• 2015 - 2019,  Professor of Biomedical Sciences, College of Medicine, Roseman University.

• 2016 - present,  Member of the Board of Directors, Nevada Consortium for Dementia Research (NCDR), state-wide consortium advocating the increasing of research funding & research opportunities/activities for developing new treatments and new biomarkers for all forms of dementia, with special emphasis on Alzheimer's disease.

Special research accomplishments  &  21 years of service as  Assistant,  Associate  &  Full Professor (medical student & graduate student teaching & mentoring medical research projects) at three Medical Schools  ( Loyola University of Chicago, Stritch School of Medicine,  University of Kentucky College of Medicine  &  The Chinese University of Hong Kong, Faculty of Medicine ) :

Early research training & early discoveries by Dr. Fiscus (physiological, pharmacological & pathological role of Nitric Oxide (NO) & its activations of the cGMP - Protein Kinase G (PKG) signaling pathway in cardiovascular and neural cells) ended up contributing to the 1998 Nobel Prize in Physiology or Medicine.  This research occurred while Dr. Fiscus served as a Senior Postdoctoral Fellow of the Am. Heart Assoc.  and then as a Fellow in Clinical Pharmacology and Scientist at Stanford University School of Medicine & Palo Alto VA Medical Center.  At Stanford  Medical School, Dr. Fiscus was mentored by Dr. Ferid Murad (1998 Nobel Prize Recipient), Associate Chair of the Department of Medicine, Stanford Univ., and Chief of Medicine, Palo Alto VA Medical Center.   Of special note, Dr. Fiscus' research at Stanford University was the first to show that NO donors (e.g. sodium nitroprusside) & the newly-discovered cardiac hormone, atrial natriuretic peptide (ANP), cause vasodilation via enchanced intracellular catalytic/kinase activity of PKG in blood vessels (Fiscus, Rapaport & Murad, 1983-1984;  Fiscus, Rapaport, Waldman & Murad, 1985;  Fiscus and Murad, 1988;  Fiscus, 1988, Review of the involvement of NO, produced by eNOS in vascular endothelial cells, in regulating the cardiovascular system, maintaining healthy levels of blood pressure and blood flow).

These early studies by Dr. Fiscus at Stanford University Medical School during the mid-1980s were also the first to show that even basal release of endogenously-produced NO (synthesized by eNOS in healthy vascular endothelial cells, and called EDRF at the time) was sufficient to modestly (but significantly) stimulate the catalytic/kinase activity of PKG in arteries, resulting in a tonic/sustained activation of PKG that protected these blood vessels against excessive vasoconstriction.  This provided the early evidence that basal release of healthy-level NO (e.g. picomole to very low nanomole levels) produced by eNOS in endothelial cells (if cells are healthy & undamaged by traumatic injury or chronic inflammation of diabetes), via its downstream activation of PKG, could provide a molecular mechanism for preventing hypertension and other cardiovascular diseases.  

Prevention of other pathologies, including Alzheimer's disease (AD), erectile dysfunction (ED) and type-1/type-2 diabetes are now known to involve this healthy-level release of NO from endothelial cells (i.e. from eNOS-containing cells in close proximity to brain neurons, penile-erectile cells and pancreatic beta-cells), via downstream activation of PKG in the protected target cells.  More recent data from the R.R. Fiscus Lab have now shown that these protective/disease-preventing effects of PKG specifically involve one isoform of PKG, i.e. the PKG1alpha splice variant, which phosphorylates key downstream target proteins (e.g. BAD, CREB, c-Src, GSK-3-beta, NF-kappaB, RhoA and VASP) mediating the anti-inflammatory, anti-oxidative stress and anti-nitrosative stress effects of healthy-level NO/cGMP/PKG-I-alpha signaling.  (see Model below, Models in the Research section and the Publications section)

Dr. Fiscus then served as an Assistant Professor in the Department of Physiology, Loyola University of Chicago, Stritch School of Medicine, teaching medical students and setting up/supervising the department's cell culture lab.  Key studies by Dr. Fiscus at that time showed that selective inhibitors of phosphodiesterase type-5 (PDE-5 inhibitors, the prototypes of Viagra, Cialis and Levitra) could be used as a safer alternative to using NO donors (which are often toxic) and could be used to synergistically enhance the vasodilatory effects of the cardiac hormones, atrial natriuertic peptide (ANP) and brain (B-type) natriuretic peptide (BNP) (Zhou, H.L. and Fiscus, R.R., 1989). 

Dr. Fiscus was then recruited by Dr. William Markesbery, Director of the Sander-Brown Center on Aging in the University of Kentucky, College of Medicine, to work on research projects in collaboration with Dr. Mark Mattson, studying Alzheimer's related proteins and peptides, including the amyloid plaque-producing peptide, A-beta, and its precursor, APP (amyloid precursor protein).   We found that the secreted form of APP, i.e. sAPP-alpha, had neuroprotective effects, preventing the neurotoxicity & excess intracellular calcium caused by excess/pathological-level glutamate, effects that are opposite to those of the smaller fragment peptide, A-beta (which was originally thought to be as major cause of Alzheimer's disease).  We showed that sAPP-alpha's neuroprotective effects were mediated by its ability to activate a cell-surface receptor with particulate guanylyl cyclase activity (very similar to ANP and BNP receptors), elevating cGMP levels and increasing PKG kinase activity in hippocampal/memory-associated neurons (Barger, Fiscus,  Ruth, Hoffman & Mattson, 1995).  

This established an essential role of PKG catalytic/kinase activity in protecting memory-associated neurons of the brain from the neurotoxic effects of excess glutamate (as occurs in the pathogenesis of Alzheimer's disease).   These studies from the Mattson lab and Fiscus lab at Sanders-Brown Center on Aging during the mid-1990s provided evidence for a new hypothesis about the pathology of Alzheimer's disease, i.e. that the neuronal damage and loss of healthy synaptic function that cause loss of memory in the brain of Alzheimer's patients involve not only the neurotoxicity of excess A-beta production, but, importantly, also the pathological switch away from production of the healthy APP product, i.e. the neuroprotective sAPP-alpha, toward the production of neurotoxic A-beta.   We reasoned that this pathological switch from sAPP-alpha to A-beta would result in a loss of the neuroprotective cGMP/PKG activation that is normally induced by sAPP-alpha in the healthy brain.   

Production and secretion of sAPP-alpha and the activation of the cGMP/PKG signaling pathway are both dramatically reduced in the brain of Alzheimer's patients (Habib, Sawmiller & Tan, Restoring soluble amyloid precursor protein alpha functions as a potential treatment for Alzheimer's disease, 2017;  Rosenberger AFN et al., Protein kinase activity decreases with higher Braak stages in Alzheimer's disease pathology, 2016 - article showing dramatically reduced phosphorylation of PKG-specific downstream target proteins, such as phosphodiesterase type-5A and VASP-serine-239, indicting a dramatic decrease in the catalytic/kinase activity of PKG in the brains of Alzheimer's patients with higher Braak stages).

Following  Sanders-Brown Center on Aging and the University of Kentunky College of Medicine, Dr. Fiscus was then recruited to a new medical school in Hong Kong, the Chinese University of Hong Kong (CUHK), noted for its emphasis and support for quality medical research.   Dr. Fiscus served as Full Professor in the Department of Physiology, Faculty of Medicine, and as the Head of the Molecular Gerontology section (including heading research on the Molecular Pathology of Alzheimer's disease, Cardiovascular diseases, Diabetes and Ovarian Cancer and the discovery of shared molecular aberrations involved in all of these pathologies) within the Center for Gerontology & Geriatrics at CUHK.  Dr. Fiscus also served as the Molecular Pharmacologist for CUHK's Center for Chinese Medicine.  Specifically,  Dr. Fiscus' lab studied the molecular mechanisms of action of various herbal medicines, including ginseng and the tannins of Geum japonicum, showing that both are capable to lowering the blood pressure in hypertensive animal models via a mechanism involving the cardiovascular release of NO and the downstream activation of the cyclic GMP signaling pathway in blood vessels.

Administrative Experience as Director of Cancer Molecular Biology Section at Nevada Cancer Institute (NVCI) & the first Vice President for Research (university-wide:  Colleges of Pharmacy, Dental Medicine & Nursing) for Roseman University of Health Sciences  &  first Associate Dean of Research for Roseman University's new College of Medicine:

Dr. Fiscus was then recruited in 2006 to become the first Director of the Cancer Molecular Biology Section at the newly-opened Nevada Cancer Institute (NVCI) in the Summerlin (far-western, mountain's-edge) district of metropolitan Las Vegas, Nevada, USA, with research emphasis on the discovery of novel protein kinases involved in mediating the exaggerated proliferation and resistance to chemotherapy (chemoresistance) in lung cancer, ovarian cancer and prostate cancer.  We found that each of these cancers possessed a pathological/uncontrolled hyperactivation of the PKG1alpha splice variant that mediated the exaggerated proliferation and chemoresistance.  We also found that CD44, a cell-surface protein involved in adhesion to the extracellular matrix proteins and receptor for osteopontine and hyaluronic acid, provided the best biomarker for identifying the cancer stem cell (CSC) population (i.e. lung cancer cells possessing high expression levels of the pluripotency gene products Oct4, Sox2 and Nanog & exaggerated tumor-promoting activity) in lung cancer cell lines (10 established lung cancer cell lines) and within 141 tumor biopsies from lung cancer patients (see Leung, Fiscus et al., 2010 in Publications section).

In 2010, Dr. Fiscus was then recruited to Roseman University of Health Sciences, with campuses in Henderson, NV and South Jordan, UT, to oversee & give guidance for their medical & pharmaceutical research programs in the Colleges of Pharmacy, Dental Medicine & Nursing.  Dr. Fiscus served as Vice President for Research (university-wide) and Director of the new Diabetes & Obesity Research Program and specifically lead the effort to establish research activities for Roseman University in a Summerlin-Las Vegas, NV location, a site that eventually became Roseman's new Summerlin Campus for their new College of Medicine.  Dr. Fiscus then served as the first Associate Dean of Research for the College of Medicine., Roseman University.   

Research Interest

The Fiscus Lab has pioneered the development and novel uses of advanced proteomic technologies, including the new ultrasensitive near-infrared-fluorescence (NIRF)-based kinase assays (developed in the Fiscus Lab, see Patent application below), which has clear advantages over the older hazardous radioactivity-based kinase assays (see discussion below in Expertise), and a new exquisitely-sensitive robotic capillary isoelectric focusing (cIEF) technology, the NanoPro-100 & NanoPro-1000 systems (below in Expertise), to study the novel biological roles of known proteins and to identify the roles of previously-unrecognized/lower-abundance proteins (e.g. PKG1alpha) that are involved in protecting the survival and function of healthy cells (see Model on the right and the Models in Research section and our Publications).   

Our special focus has been on discovering the novel role of PKG1alpha (and its interdependence with Akt and c-Src) in various pathologies, including Alzheimer's disease (AD), type-1/type-2 diabetes (T1D & T2D), erectile dysfunction (ED), cardiovascular diseases (CVDs) and certain cancers (brain, breast, lung, ovarian, pancreatic and prostate cancers).   The exaggerated cell proliferation and cell survival (causing resistance to chemotherapy, i.e. chemoresistance) in these cancer cells involves the pathological high levels of kinase activity (i.e. hyperactivation) of PKG1alpha.  The R.R. Fiscus Lab has found that  reciprocol  phosphorylation  &  activation  with c-Src, a tyrosine kinase that can serve as an oncogenic protein when over-expressed or hyperactivated (typical for most cancers), occurs in various cancer cells, resulting in the hyperactivation of PKG1alpha.  In contrast, diabetes and insulin resistance-related pathologies (e.g. AD, ED and CVD) typically involve pathologically low (inadequate) levels of PKG1alpha kinase activity, resulting in loss of cell function and damaged cell survival (see Model on the right and Models in the Research section as well as Publications section).

Our current research focuses on three types of cells (the memory-processing/storing neurons of the brain, the insulin-secreting pancreatic beta-cells and the vascular endothelial cells) that are among the most vulnerable cells to damage caused by excessive inflammation, oxidative stress and nitrosative stress.  Nitrosative stress involves damage of proteins caused by excessive accumulation of the toxic forms of NO (e.g. peroxynitrite, formed from the chemical combination of superoxide and high-level NO resulting from  pathological hyper-expression  of iNOS or from  pathological  uncoupling  of eNOS and nNOS), which mediates abnormal S-nitrosylation and tyrosine-nitration of proteins, damaging their normal function and resulting in loss of cell functions (e.g. causing synaptic dysfunction in brain neurons) and ultimately cell death.  We have identified the PKG1alpha splice variant as a key  cell-survival protein  that enhances the activation of several insulin signaling proteins (e.g. PI3-kinase and Akt), protecting against insulin resistance during obesity and metabolic syndrome, and preserves healthy function of cells by stopping and reversing the cellular damage caused by excessive inflammation, oxidative stress and nitrosative stress.  PKG1alpha is the protein kinase that is activated by healthy-level/healthy-form NO [produced by eNOS (in vascular endothelial cells and several other types of cells, e.g. epithelial cells) and nNOS (in neural cells and skeletal muscle cells)] and by healthy-level carbon monoxide (CO) from heme oxygenase (HO-1 or HO-2). 

The three types of vulnerable cells studied in the Fiscus Lab are:  1) the memory-storing neurons of the brain, involved in Alzheimer's disease,  2) the insulin-secreting beta-cells in pancreatic islets, involved in both type-1 & type-2 diabetes, and  3) the vascular endothelial cells lining blood vessels that normally regulate blood pressure, blood flow and the delivery of oxygen and nutrients to all organ systems, involved in many complications of diabetes, including cardiovascular damage (e.g. hypertension, atherosclerosis), kidney damage and retinal damage (potentially resulting in blindness) caused by the chronic inflammation, oxidative stress and nitrosative stress of type-1/type-2 diabetes.  

In neural cells, pancreatic beta-cells and endothelial cells [and also less vulnerable cells, e.g. bone marrow-derived mesenchymal stem cells (BM-MSCs) and certain types of cancer cells, i.e. brain, lung & ovarian cancers], the R.R. Fiscus Lab has identified a slice variant/isoform of protein kinase G (PKG), PKG1alpha, that plays an essential role in promoting increased cell survival and regeneration by increasing the phosphorylation of key downstream target proteins, Akt/FoxO1, BAD, CREB, c-Src and VASP, which enhance the expression of cell-survival proteins, including Bcl-2 family members (e.g. Mcl-1) and Inhibitor of Apoptosis Protein (IAP) family members (e.g. c-IAP1, c-IAP2, Livin, Survivin & , in some cells, XIAP) and anti-oxidant/anti-inflammatory & anti-nitrosative-stress proteins (thioredoxin, TRX, capable of reversing pathological S-nitrosylation) to protect these cells against the damage from prolonged/excessive inflammation and oxidative/nitrosative stress (see Publications).  

The R.R. Fiscus Lab currently studies the role of PKG1alpha and its inter-connection (cross-talk) with several other protein kinases (e.g. Akt, GSK-3-beta, RhoA/ROCK1/2 & c-Src) in mediating:   (1) protection of neural cell survival and function, helping to prevent neurodegenerative diseases such as Alzheimer's, Parkinson's, and (potentially) Lewy body dementia (LBD),  (2) improved survival and regeneration of pancreatic beta-cells for protection against type-1 and type-2 diabetes and improving the survival and function of beta-cells during islet transplantation therapy (see Wong, Vo, Gorjala & Fiscus, 2017, listed in Publications), and  (3) exaggerated cancer cell proliferation and resistance to chemotherapy (chemoresistance) caused by abnormal/pathological interactions, e.g. excessive reciprocol phosphorylation & activation between PKG1alpha (a serine/threonine kinase) and c-Src (a tyrosine kinase with oncogenic potential) that can cause pathological-level/uncontrolled hyperactivation of PKG1alpha & exaggerated over-production of the cell-survival proteins Mcl-1, c-IAP1, Livin and Survivin (see Wong & Fiscus, 2012, see Publications).

The R.R. Fiscus Lab also studies Resveratrol (a medicinal polyphenol in red wine, grapes, berries, peanuts), the tannins from Geum japonicum & other plant-derived medicinal/nutraceutical chemicals from foods, drinks & herbal medicines, determining the cytoprotective/anti-oxidant/anti-inflammatory effects and their potential for preventing and reversing the pathology of Alzheimer's, Parkinson's, cardiovascular diseases, diabetes, ED & some cancers.  We are particularly interesting in determining novel molecular mechanisms of action (MOAs) of these medicinal chemicals, using our advanced, exquisitely-sensitive proteomic technologies for discovery of novel proteins, isoforms/splice variants & PTMs and protein-protein interdependence of these chemicals to identify new molecular targets for prevention and effective treatment of diseases.  

Our current studies build upon our earlier findings showing that tannins (complex polyphenols) of Geum japonicum cause vasorelaxation and anti-hypertensive effects in spontaneously hypertensive animal models via the tannins' ability to stimulate endothelial cell production and release of (cardiovascular-protective) NO and elevation of cGMP levels in smooth muscle cells of blood vessels (Xie, Xu, Dong, Fiscus and But, 2007).   It is anticipated that these tannins may have similar protective effects in neural cells (protecting against Alzheimer's and other neurodegenerative conditions) and pancreatic beta-cells (protecting against type-1/type-2 diabetes), thus helping to protect against the damage of these cells during excessive inflammation and oxidative/nitrosative stress. 

Our more recent studies with the polyphenol resveratrol have shown that RESVERATROL, at lower concentrations, can activate PKG1alpha in human ENDOTHELIAL CELLS (via upregulation of eNOS expression/activity and subsequent increased production of healthy-level NO), which improves endothelial cell survival and proliferation (i.e. healthy angiogenesis), a mechanism that can promote wound healing & tissue regeneration (Wong & Fiscus, 2015 (research article) & Wong, Gorjala, Costatino & Fiscus, 2016 (book chapter), see Publications).  In contrast, we also found that higher/anti-cancer concentrations of resveratrol cause inhibition of human endothelial cell survival and proliferation (anti-angiogenesis effects) via the ability of higher-level resveratrol to directly inhibit PKG1alpha catalytic/kinase activity.  Our studies showed that direct inhibition of PKG1alpha kinase activity by anti-cancer-levels of resveratrol cause dramatic decreases in the protein expression levels of several of the Inhibitor of Apoptosis Proteins (IAPs), including c-IAP1, Livin, Survivin & XIAP in human endothelial cells  (Wong & Fiscus, 2015 &  Wong, Gorjala, Costatino & Fiscus, 2016 book chapter, see Publications).

Expertise

In 2013, we set up the second, even more versatile NanoPro proteomic system, the NanoPro-1000, for research in the fields of Neuroscience (e.g. helping to identify the PKG1alpha  splice variant as an essential protein kinase in promoting survival of neural cells) and Endocrinology/Metabolism [e.g. identifying the PKG1alpha splice variant (and its interdependence with insulin signaling proteins such as Akt/FoxO1) as an essential protein kinase in mediating survival and proliferation (regeneration) of pancreatic beta-cells (Wong, Vo, Gorjala & Fiscus, 2017) and the PKG-I "SPLICE VARIANT SWITCH" that occurs during fat cell differentiation] (Johlfs et al., PLoS One, 2015) in Publications section.  As an example of  isoform/splice-variant switching , we found that healthy (non-inflammatory) pre-adipocytes  express predominately the PKG1alpha splice variant of PKG-I, whereas the lipid-droplet-engorged, pro-inflammatory fat cells of these adipocytes, after differentiation, express predominately the alternative splice variant, the PKG1beta, but with dramatically reduced expression of the PKG1alpha splice variant.  (see Publications, Johlfs et al., PLoS One, 2015)

This isoform switch, from predominantly the cytoprotective PKG1alpha isoform to predominantly the PKG1beta isoform, during pre-adipocyte differentiation gives us clues about the roles of these two isoforms in the regulation of cell function and survival.  PKG1alpha, as a cell-survival protein that enhances the expression of anti-oxidant and anti-inflammatory proteins, helps to protect the pre-adipocytes from oxidative stress and prevents inflammation - the anti-inflammatory actions of PKG1alpha likely involves the suppression of the pro-inflammatory form of NF-kappaB that leads to  production of pro-inflammatory cytokine, IL-1-beta, IL-6 and TNF-alpha (see Models in the Research section).   However, during differentiation and lipid-droplet accumulation, fat cells dramatically reduce their expression of the cytoprotective PKG1alpha isoform, resulting in loss of the anti-oxidant and anti-inflammatory effects of this protein kinase splice variant.  Fat-engorged adipocytes, because of their dramatically reduced expression of PKG1alpha, become damaged because of losing this protective mechanism,  with resulting mitochondrial damage and increased expression of pro-inflammatory cytokines (like that occurring during the pathogenesis of obesity).  

Recent research articles and reviews from Francis Kim's lab at the University of Washington-Seattle and his collaborators in Woo Je Lee's lab at University of Ulsan College of Medicine, Seoul, South Korea,  have shown that PKG-I, via its ability to phosphorylate VASP at serine-239, inhibits the pro-inflammatory form of NF-kappaB and thereby inhibits the pro-inflammatory phenotype of various cells, e.g. vascular cells, liver cells and fat cells, and reduces the production of pro-inflammatory cytokines that typically are produced in animals given a high-fat diet.   

From our data, it appears that this anti-inflammatory mechanism of PKG-I, i.e. preventing high-fat-diet/obesity-induced chronic inflammation, likely depends of which splice variant of PKG-I is being expressed.  Thus, the cell-differentiation-induced switch of PKG-I splice variant expression in fat cells likely contributes to the chronic inflammation and insulin resistance occurring during the pathogenesis of obesity and type-2 diabetes.

The Fiscus Lab has also collaborated with the Timothy T. Le lab at Roseman University's Summerlin Campus, highlighting the research of Dr. Yasuyo Urasaki, to further utilize the advanced robotic cIEF proteomic technology (the NanoPro-1000 system), this time to assess liver tissue abnormalities associated with three types of fatty liver disease [high-fat diet (HFD)-induced,  drug-therapy-induced and genetic mutation-induced fat accumulation and damage of the liver].  

The NanoPro-1000 was especially well-adapted at identifying the differences in three PTMs (acetylation, glycosylation and phosphorylation) of a panel of 12 selected proteins (metabolic and cell signaling proteins), illustrating the exceptional value of this new cIEF technology for identifying proteomic changes (proteomic profiling) that could be used for effectively diagnosing the differences in these three types of liver pathologies (see Publications, Urasaki, Fiscus & Le, Journal of Pathology 2016) and Patent Application.  

Patent Application WO 2017-035323-A1 - Published Mar. 2, 2017, Inventors: Timothy Thuc Le, Yasuyo Urasaki & Ronald R. Fiscus. for use in diagnosing Fatty Liver Diseases)  

Our recent publications illustrate the exciting potential of using this exquisitely-sensitive technology of NanoPro-1000 (robotic cIEF) as a diagnostic tool, identifying key biomarkers of various diseases, to help differentiate between different forms/different causes of these diseases, including type-1 diabetes versus type-2 diabetes, Alzheimer's disease versus other forms of neurological diseases and the various forms of cancer.   

The novel applications of this new technology in the R.R. Fiscus Lab and collaborators at Roseman University of Health Sciences have been defined in our recent publications:   

1.  Fiscus & Johlfs, 2012, book chapter in Protein Kinase Technologies, in the series NEUROMETHODS ;   

2.  Fiscus et al., 2012, book chapter in Ovarian Cancer - Basic Science Perspectives;   

3.  Johlfs, Gorjala, Urasaki, Le and Fiscus, 2015, research article in PLoS One;   

4.  Wong  & Fiscus, 2015, research article in AntiCancer Research;   

5.  Wong, Gorjala, Costantino & Fiscus, 2016, book chapter in Gynecologic Cancers - Basic Sciences, Clinical and Therapeutic Perspectives;  

6.  Urasaki, Fiscus & Le, 2016, research article in Journal of Pathology, with Invited Commentary by Frances L. Gyrne & Kyle L. Hoehn (experts on Pathological Diagnosis),  "Subclassification of fatty liver by its pathogenesis:  cIEFing is believing", highlighting the exceptional value of this new exquisitely-sensitive and versatile technology in the diagnosis of fatty liver diseases and many other diseases.   

7.  Wong, Vo, Gorjala & Fiscus, 2017, research article in Diabetes & Vascular Disease Research.  (identifying PKG1alpha as a key "cell survival protein" and a  novel protein kinase that promotes insulin signaling, e.g. enhanced activation of Akt, downstream phosphorylation of CREB and FoxO1 and expression of PDX-1, in pancreatic beta-cells), which promotes beta-cell survival and proliferation/regeneration (see Publications).

Newly-developed Near-InfraRed-Fluorescence (NIRF)-based kinase assay technology for ultrasensitive measurements of the catalytic/kinase activity of any protein kinase in biological or clinical samples, conducted in a rapid, non-radioactive (safer) manner (developed in the R.R. Fiscus Lab). 

This new technology uses selective protein kinase substrates and selective protein kinase inhibitors to define a specific protein kinase catalytic activity within a complex mixture of protein kinases, such as biological samples (tissue homogenates, cell culture lysates) or clinical samples (blood, cerebrospinal fluid, urine, saliva, etc.), designed for the development of new therapeutic agents and for the discovery of new disease biomarkers, based on the catalytic/kinase activity (functional activity) of novel target proteins. 

(see our Patent Application US 2014/0287447-A1 - Published Sept. 25,  2014,  Inventors:  R.R. Fiscus, B. Costantino, M.G. Johlfs & J.C. Wong).  

The near-infrared-fluorescence (NIRF)-based protein kinase assays provide a safer alternative to the hazardous older methods of measuring protein kinase catalytic activity using P-32 or P-33 radioactivity.  Also, unlike another commonly-used technology, i.e. microchip-electrophoresis-based kinase assays, which require exclusively the use of protein kinases that have been "cleaned up" as either "recombinant proteins" or biological samples that have been extensively purified (to remove other proteins that would interfere with the analysis), our new NIRF-based technology allows for the immediate measurements of kinase/catalytic activity of any specific protein kinase [e.g. the AGC kinases (PKA, PKG & PKC), GSK-3-beta,  or any protein kinase) within a complex mixtures of proteins, such as tissue homogenates, cell lysates and liquid biopsies (blood, cerebrospinal fluid, urine, saliva, etc.) without the need of sample "clean up" that is time-consuming and potentially damaging to the measurement of kinase activity.  Our experience has shown that delays of even a couple minutes (from the time of collecting samples to the beginning of the kinase activity measurements) can result in dramatic changes in the measured catalytic activities, thus giving false information about kinase activity levels.  

Examples of TIRF microscopy at Roseman University of Health Sciences, compared side-by-side with conventional confocal microscopy, using the TIRF/confocal imaging of the same cells, in this case, the H1299 lung cancer cells.  In 2010, the R.R. Fiscus Lab identified the H1299 cells as possessing a very high percentage (>80%) of cells with cancer stem cell (CSC) characteristics, i.e. high-level expression of the pluripotency gene products, Oct4, Nanog and Sox2 (Leung et al., 2010, see Publications section).  Our 2010 study identified CD44, a cell-surface protein previously used to help identify CSCs in breast and prostate cancer, as the  best  Biomarker  for CSCs in lung cancer, both in cell lines (analyzing 10 non-small cell lung cancer cell lines) and in clinical samples of 141 resected lung tumors (Leung et al., 2010).  

The TIRF-versus-confocal images show the clear advantages of TIRF microscopy, a technology that illuminates & visualizes only 100 nm into attached cells, thus eliminating approximately 99% of the fluorescence background noise.  This allows labeled proteins (at the plasma membrane and closely associated at the membrane inner surface) to be imaged with a 100-fold improvement in the signal-to-noise ratio, thus potentially giving a 100-fold better sensitivity compared with other fluorescence microscopy techniques.   This then allows for the effective imaging of lower-abundance proteins, such as receptors and signaling proteins, that are localized at the plasma membrane (especially at attachment sites) but not widely distributed throughout the cytosol. 

Note the difficulty in imaging c-Src and PKG-I by confocal microscopy (because of the lower abundance of these proteins within the cytosol of lung cancer cells), but clear imaging of c-Src and PKG-I at the plasma membrane using TIRF microscopy, which images only the first 100 nanometers closest to the plasma membrane of the cells.  Importantly, TIRF is able to show the close association (co-localization) of c-Src and PKG-I with the stem cell biomarker CD44 (shown in yellow/orange color in the merged TIRF images), all three proteins (CD44, c-Src and PKG-I-alpha) present at the plasma membrane.  

Previously, we found that PKG-I-alpha and c-Src directly interact, in a previously-unrecognized way, in which PKG-I-alpha phosphorylates c-Src at serine-17 (enhancing c-Src's tyrosine kinase activity) and c-Src tyrosine-phosphorylates PKG-I-alpha (tyrosine site unknown), increasing the serine/threonine kinase activity of PKG-I-alpha.  This "reciprocal phosphorylation/activation" between PKG-I-alpha and c-Src is illustrated in our Models in the Research section, and is described in other publications, see Publications section.

While serving as Vice President for Research for Roseman University of Health Sciences, Dr. Fiscus, in collaboration with Mary G. Johlfs, Roseman's Director of Research Operations - Summerlin Campus, had set up the first TIRF microscopy imaging system in Southern Nevada several years ago.  To improve the utility of this system, we coupled this TIRF system with a conventional confocal microscopy system using the same microscope base, thus allowing the imaging and analysis of the same cells using both confocal and TIRF microscopy technologies within the same sample (at the flip of a switch).

Confocal can be used to generate optical sections (typically 0.5 - 1 micron in thickness) of images of fluorescent-labeled cells, with the potential of generating 3-D images and showing the subcellular localization of any labeled protein.  However, a disadvantage with confocal microscopy, like most other fluorescence-based microscopy methods, is the potential of background noise (e.g. autofluorescence), which can limit the ability to image certain proteins, especially lower-abundance proteins (like some signaling proteins that localize at membrane, rather than being evenly distributed throughout the cytosol).  Key signaling proteins  (like PKG-I-alpha and c-Src) are often missed (and overlooked/unrecognized) because of this problem.

TIRF microscopy, in contrast, provides a mechanism of eliminating 99% of the background noise, thus giving a 100-fold increase in the signal-to-noise ratio (i.e. 100-fold higher sensitivity) and allowing the imaging of lower-abundance proteins (especially plasma membrane-associated proteins).  The special laser-mediated illumination system provided by the TIRF technology (i.e. bouncing a laser beam of light off the bottom of a glass slide at just the right angle to generating evanescence waves that enter the cells by only 100 nanometer) results in the illumination of only those proteins closely associated with the plasma membrane, and eliminating 99% of the background noise that is typically present in the rest of the cell.  The TIRF technology provides a very thin slice of imaging of the cell, only 100 nanometers (0.1 microns) in thichness, a much thinner slice than achieved by confocal microscopy, thus eliminating 99% of the background noise and improving sensitivity by about 100-fold.  The TIRF technology provides clear advantages for studying any protein localized at the plasma membrane (cell surface proteins, transmembrane proteins & intracellular proteins attached to the internal surface of the plasma membrane), like the stem cell biomarker CD-44 and the key cell-survival-regulatory protein kinases c-Src and PKG-I-alpha.