Ashraf M. Mohieldin

Contact:      
Mohieldi@chapman.edu

EDUCATION:
• Post-Doctoral Fellow, February 2015 to present, School of Pharmacy, Chapman, University, CA, USA.
• Ph.D. Degree in Medicinal and Biological Chemistry, August 2015, School of Pharmacy, The University of Toledo, OH, USA
• B.S. Degree in Pharmaceutical science, May 2011, College of Pharmacy, University of, Toledo, OH, USA.

RESEARCH EXPERIENCE:
• Ph.D. dissertation, University of Toledo, Medical center, Toledo, OH – Surya M. Nauli, PhD, August 2011 to August 2015.
• Research Assistant, University of Toledo, Medical center, Toledo, OH – Surya M. Nauli, PhD, May 2011 to August 2011.
• Departmental Honor Thesis (Pharmacology), University of Toledo, Medical center, Toledo, OH – Surya M. Nauli, PhD, January 2010 to May 2011.
• Research Assistant, University of Toledo, Medical center, Toledo, OH – John J. Bellizzi, PhD, January 2009 to May 2009.
• Research Assistant, University Of Massachusetts Boston, Boston, MA – Kenneth Campbell, PhD, May 2008 to August 2008.
• Lab Assistant, University of Toledo, Toledo, OH – Julie Mosher, Chemical Supervisor, August 2007 to May 2008.
• Lab Assistant, Bunker Hill College, Cambridge, MA – John Steeper, PhD, May 2005 to May 2007.

PHRAMACY PRACTICE EXPERIENCE:
• Community pharmacy, Rite Aid Pharmacy, Toledo, OH – Elizabeth Miller, pharmacist, January 2015 to December 2015.
• Clinical shadowing P1 year- University of Toledo, Medical center, Toledo, OH, Michelle Schroeder, PharmD, March 2015.

RESEARCH WORK:
• Designed new approach to mechanically induced ciliary protein compositions by using a sensitive multidimensional protein identification technology (MudPIT) for analysis, Chapman University, CA, USA.
• Designed the work on combining high-pressure freezing (HPF) and freeze-fracture transmission electron microscopy (FFTEM) technique to develop an optimal highresolution imaging approach that preserves cilia structures in their best natural form without alteration of cilia morphology by chemical fixation interference, Chapman University, CA, USA.
• Investigated the primary cilia membrane swellings (bulbs) and demonstrated for the first time their unique ultrastructure, motility under shear stress and identified key ciliary
proteins localized on them, The University of Toledo, OH, USA.
• Participated in an integrated single-cell imaging technique project and demonstrated for the
first time that calcium signaling triggered by fluid-shear stress initiates in the primary cilium and can be distinguished from the subsequent cytosolic calcium response through the ryanodine receptor, The University of Toledo, OH, USA.
• Participated in a clinical study in PKD patients where our crossover, multicenter, doubleblind and placebo-controlled clinical study further indicated that cilia-targeting therapy showed an overall reduction in mean arterial pressure in PKD patients, The University of Toledo, OH, USA.
• Investigated the dynamic function of primary cilia in normal condition and under fluidshear stress, along with the function and the contents of ciliary bulb with and without Nocodazole drug, The University of Toledo, OH, USA.
• Designed new approach to study primary cilia from the side by using flexible substratum, The University of Toledo, OH, USA.
• Studied many proteins that involved in regulating the circadian rhythms, The University of Toledo, OH, USA.
• Worked on optimizing DNA Detection Sensitivity by lowering the detection limit to femtomolar by constructing newly build Spectrofluorometer instrument, University of Boston Massachusetts, MA, USA.

PROFESSIONAL AWARDS:
• NIH F31 fellowship award, University of Toledo, Medical center, Toledo, OH, 2012-2015
• Departmental Honor Thesis, University of Toledo, Medical center, Toledo, OH, May 2011
• Dean’s list, University of Toledo, Medical center, Toledo, OH, spring 2011
• Dean’s list, University of Toledo, Medical center, Toledo, OH, spring 2009
• Dean’s list, University of Toledo, Medical center, Toledo, OH, Fall 2008
• Dean’s list, University of Toledo, Medical center, Toledo, OH, spring 2009

PROFESSIONAL MEETINGS:
• Mohieldin, A.M. et al. Dynamics structures of ciliary length and bulbs are mechanically
regulated [abstract]. Keystone symposia on molecular and cellular biology conference, Cilia development and human diseases, Tahoe, California (2013).
• Mohieldin, A.M. et al. Protein composition and movements of membrane swellings
associated with primary cilia [abstract]. Gordon research conference. Cilia, Mucus & Mucociliary Interactions, Galveston TX (2015).

BOOK CHAPTER PUBLICATIONS:
• Mohieldin, A.M. et al. Autosomal Dominant Polycystic Kidney Disease: Pathophysiology and Treatment. Nova Publishers. c2013 [ISBN: 978-1-62808-760-4]

SUMMARY OF PUBLICATIONS:

Doerr, N., et al. (2016). “Regulation of Polycystin-1 Function by Calmodulin Binding.” PLoS One 11(8): e0161525.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM.

 

Jin, X., et al. (2014). “Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli.” Cell Mol Life Sci 71(11): 2165-2178.

Primary cilia with a diameter of ~200 nm have been implicated in development and disease. Calcium signaling within a primary cilium has never been directly visualized and has therefore remained a speculation. Fluid-shear stress and dopamine receptor type-5 (DR5) agonist are among the few stimuli that require cilia for intracellular calcium signal transduction. However, it is not known if these stimuli initiate calcium signaling within the cilium or if the calcium signal originates in the cytoplasm. Using an integrated single-cell imaging technique, we demonstrate for the first time that calcium signaling triggered by fluid-shear stress initiates in the primary cilium and can be distinguished from the subsequent cytosolic calcium response through the ryanodine receptor. Importantly, this flow-induced calcium signaling depends on the ciliary polycystin-2 calcium channel. While DR5-specific agonist induces calcium signaling mainly in the cilioplasm via ciliary CaV1.2, thrombin specifically induces cytosolic calcium signaling through the IP3 receptor. Furthermore, a non-specific calcium ionophore triggers both ciliary and cytosolic calcium responses. We suggest that cilia not only act as sensory organelles but also function as calcium signaling compartments. Cilium-dependent signaling can spread to the cytoplasm or be contained within the cilioplasm. Our study thus provides the first model to understand signaling within the cilioplasm of a living cell.

 

Kathem, S. H., et al. (2014). “Ciliotherapy: a novel intervention in polycystic kidney disease.” J Geriatr Cardiol 11(1): 63-73.

BACKGROUND: Ciliopathies are a group of diseases associated with abnormal structure or function of primary cilia. Ciliopathies include polycystic kidney disease (PKD), a pathology associated with vascular hypertension. We previously showed that cilia length regulates cilia function, and cilia function is required for nitric oxide (NO) biosynthesis in endothelial cells. Because patients with PKD show abnormal sensory cilia function, the aim of our current study was to search for a targeted therapy focused on primary cilia, which we refer to as ‘ciliotherapy’. METHODS AND RESULTS: In the present studies, our in vitro analyses refined fenoldopam as an equipotent and more specific dopaminergic agonist to regulate cilia length and function. Our in vivo studies indicated that fenoldopam increased cilia length and serum NO thereby reducing blood pressure in a PKD mouse model. Our crossover, multicenter, double-blind and placebo-controlled clinical study further indicated that cilia-targeting therapy showed an overall reduction in mean arterial pressure in PKD patients. CONCLUSIONS: Overall, our studies provide the first evidence of ciliotherapy as an innovative intervention in patients with abnormal primary cilia.

 

Kathem, S. H., et al. (2014). “The Roles of Primary cilia in Polycystic Kidney Disease.” AIMS Mol Sci 1(1): 27-46.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited genetic disorder that results in progressive renal cyst formation with ultimate loss of renal function and other systemic disorders. These systemic disorders include abnormalities in cardiovascular, portal, pancreatic and gastrointestinal systems. ADPKD is considered to be among the ciliopathy diseases due to the association with abnormal primary cilia function. In order to understand the full course of primary cilia and its association with ADPKD, the structure, functions and role of primary cilia have been meticulously investigated. As a result, the focus on primary cilia has emerged to support the vital roles of primary cilia in ADPKD. The primary cilia have been shown to have not only a mechanosensory function but also a chemosensory function. Both structural and functional defects in primary cilia result in cystic kidney disease and vascular hypertension. Thus, the mechanosenory and chemosensory functions will be analyzed in regards to ADPKD.

 

Mohieldin, A. M., et al. (2015). “Chemical-Free Technique to Study the Ultrastructure of Primary Cilium.” Sci Rep 5: 15982.

A primary cilium is a hair-like structure with a width of approximately 200 nm. Over the past few decades, the main challenge in the study of the ultrastructure of cilia has been the high sensitivity of cilia to chemical fixation, which is required for many imaging techniques. In this report, we demonstrate a combined high-pressure freezing (HPF) and freeze-fracture transmission electron microscopy (FFTEM) technique to examine the ultrastructure of a cilium. Our objective is to develop an optimal high-resolution imaging approach that preserves cilia structures in their best natural form without alteration of cilia morphology by chemical fixation interference. Our results showed that a cilium has a swelling-like structure (termed bulb), which was previously considered a fixation artifact. The intramembrane particles observed via HPF/FFTEM indicated the presence of integral membrane proteins and soluble matrix proteins along the ciliary bulb, which is part of an integral structure within the ciliary membrane. We propose that HPF/FFTEM is an important and more suitable chemical-free method to study the ultrastructure of primary cilia.

 

Mohieldin, A. M., et al. (2015). “Protein composition and movements of membrane swellings associated with primary cilia.” Cell Mol Life Sci 72(12): 2415-2429.

Dysfunction of many ciliary proteins has been linked to a list of diseases, from cystic kidney to obesity and from hypertension to mental retardation. We previously proposed that primary cilia are unique communication organelles that function as microsensory compartments that house mechanosensory molecules. Here we report that primary cilia exhibit membrane swellings or ciliary bulbs, which based on their unique ultrastructure and motility, could be mechanically regulated by fluid-shear stress. Together with the ultrastructure analysis of the swelling, which contains monosialodihexosylganglioside (GM3), our results show that ciliary bulb has a distinctive set of functional proteins, including GM3 synthase (GM3S), bicaudal-c1 (Bicc1), and polycystin-2 (PC2). In fact, results from our cilia isolation demonstrated for the first time that GM3S and Bicc1 are members of the primary cilia proteins. Although these proteins are not required for ciliary membrane swelling formation under static condition, fluid-shear stress induced swelling formation is partially modulated by GM3S. We therefore propose that the ciliary bulb exhibits a sensory function within the mechano-ciliary structure. Overall, our studies provided an important step towards understanding the ciliary bulb function and structure.

 

Mohieldin, A. M., et al. (2016). “Vascular Endothelial Primary Cilia: Mechanosensation and Hypertension.” Curr Hypertens Rev 12(1): 57-67.

Primary cilia are sensory organelles that extend from the cell surface and sense extracellular signals. Endothelial primary cilia protruding from the inner surface of blood vessel walls sense changes in blood flow and convert this mechanosensation into an intracellular biochemical/molecular signal, which triggers a cellular response. Primary endothelial cilia dysfunction may contribute to the impairment of this response and thus be directly implicated in the development of vascular abnormalities such as hypertension and aneurysms. Using both in vitro techniques as well as in vivo animal models, we and others have investigated fluid flow mechanosensory functions of endothelial cilia in cultured cells, animal models and autosomal dominant polycystic kidney disease (ADPKD) patients. More in-depth studies directed at identification of the mechanisms of fluid flow sensing will further enhance our knowledge of cilia-dependent vascular pathology. Although the current treatments aimed at treating the cardiovascular symptoms in ADPKD patients successfully slowed the progression of cyst growth, there is growing evidence which suggests that drugs which interfere with primary cilia function or structure could reduce cardiovascular complications in ADPKD. This review is to summarize the most recent studies on primary endothelial cilia function in the vascular system and to present primary cilia as a novel therapeutic target for vascular hypertension.

 

SELECTED IMAGES:

GM3
FFTM