Joshua Slee, Ph.D

Traditionally-trained undergraduate students often lack an understanding of science as an active process that yields the information presented in their textbooks. One result has been a call for more research experiences built into traditional introductory undergraduate courses, now commonly referred to as course-based undergraduate research experiences (CUREs). The laboratory module presented in this paper used an established four-step pedagogical framework to simplify and streamline the development and implementation process of a CURE in an introductory biology laboratory setting. A unique six-week CURE was designed for undergraduates enrolled in a cell biology lab that employs Saccharomyces cerevisiae as a eukaryotic model organism. Students address a research problem that is of interest to the scientific community: Do select chemicals in the environment have adverse effects on the mitotic cell division? Students are first introduced to S. cerevisiae, its life cycle, morphology, growth curve generation and analysis, and the laboratory techniques required to cultivate this organism. Working in groups, students then act as scientists to research primary literature, ask an original question, develop a testable hypothesis, collaborate with peers, design and conduct an experiment, analyze and interpret data, and present their work to their peers. In addition, students are involved in multiple levels of iterative work, including addressing problems or inconsistencies, ruling out alternative explanations, and/or gathering additional data to support assertions. CourseSource. https://doi.org/10.24918/cs.2017.4 (2017)

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies against specific peptides within the predicted sequence allow some localization experiments. However, other possible interactions with the antibodies often cannot be excluded. This situation provided an opportunity to develop a set of assays dependent on the protein sequence. Specifically, a construct containing the gene sequence coupled to the GFP coding sequence at the C-terminal end of the protein was obtained and employed for these purposes. Experiments to characterize localization, ligand affinity, and gain of function were originally designed and carried out to confirm the identification of TMEM184A as a heparin receptor1. In addition, the construct can be employed for studies addressing membrane topology questions and detailed protein-ligand interactions. The present report presents a range of experimental protocols based on the GFP-TMEM184A construct expressed in vascular cells that could easily be adapted for other novel proteins. J Vis Exp (2017) (120), e55053, doi:10.3791/55053

The effectiveness of endovascular stents is hindered by in-stent restenosis (ISR), a secondary re-obstruction of treated arteries due to unresolved inflammation and activation of smooth muscle cells in the arterial wall. We previously demonstrated that immobilized CD47, a ubiquitously expressed transmembrane protein with an established role in immune evasion, can confer biocompatibility when appended to polymeric surfaces. In present studies, we test the hypothesis that CD47 immobilized onto metallic surfaces of stents can effectively inhibit the inflammatory response thus mitigating ISR. Recombinant CD47 (recCD47) or a peptide sequence corresponding to the Ig domain of CD47 (pepCD47), were attached to the surfaces of both 316L-grade stainless steel foils and stents using bisphosphonate coordination chemistry and thiol-based conjugation reactions to assess the anti-inflammatory properties of CD47-functionalized surfaces. Initial in vitro and ex vivo analysis demonstrated that both recCD47 and pepCD47 significantly reduced inflammatory cell attachment to steel surfaces without impeding on endothelial cell retention and expansion. Using a rat carotid stent model, we showed that pepCD47-functionalized stents prevented fibrin and platelet thrombus deposition, inhibited inflammatory cell attachment, and reduced restenosis by 30%. It is concluded that CD47-modified stent surfaces mitigate platelet and inflammatory cell attachment, thereby disrupting ISR pathophysiology. Biomaterials. (2016) http://dx.doi.org/10.1016/j.biomaterials.2016.02.008

Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparin-induced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNFα-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNFα-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity. J Biol Chem. (2016) Jan 14. pii: jbc.M115.681288.

Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells. J Biol Chem. (2016) Jan 14. pii: jbc.M115.681122.

Tissue contacting surfaces of medical devices initiate a host inflammatory response, characterized by adsorption of blood proteins and inflammatory cells triggering the release of cytokines, reactive oxygen species (ROS) and reactive nitrogen species (RNS), in an attempt to clear or isolate the foreign object from the body. This normal host response contributes to device-associated pathophysiology and addressing device biocompatibility remains an unmet need. Although widespread attempts have been made to render the device surfaces unreactive, the establishment of a completely bioinert coating has been untenable and demonstrates the need to develop strategies based upon the molecular mechanisms that define the interaction between host cells and synthetic surfaces. In this review, we discuss a family of transmembrane receptors, known as immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors, which show promise as potential targets to address aberrant biocompatibility. These receptors repress the immune response and ensure that the intensity of an immune response is appropriate for the stimuli. Particular emphasis will be placed on the known ITIM-containing receptor, Signal Regulatory Protein Alpha (SIRPhα), and its cognate ligand CD47. In addition, this review will discuss the potential of other ITIM-containing proteins as targets for addressing the aberrant biocompatibility of polymeric biomaterials. Polymers 2014; 6(10): 2526-2551; doi:10.3390/polym6102526. PMC4333742.

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits. J Vis Exp. 2014 Aug 20;(90). doi: 10.3791/51871. PMC4325827

Published data provide strong evidence that heparin treatment of proliferating vascular smooth muscle cells results in decreased signaling through the ERK pathway and decreases in cell proliferation. In addition, these changes have been shown to be mimicked by antibodies that block heparin binding to the cell surface. Here we provide evidence that the activity of protein kinase G is required for these heparin effects. Specifically, a chemical inhibitor of protein kinase G, Rp-8-pCPT-cGMS, eliminates heparin and anti-heparin receptor antibody effects on bromodeoxyuridine incorporation into growth factor stimulated cells. In addition, protein kinase G inhibitors decrease heparin effects on ERK activity, phosphorylation of the transcription factor ELK-1, and heparin induced MKP-1 synthesis. Although transient, the levels of cGMP increase in heparin treated cells. Finally, knock down of protein kinase G also significantly decreases heparin effects in growth factor activated vascular smooth muscle cells. Together, these data indicate that heparin effects on vascular smooth muscle cell proliferation depend, at least in part, on signaling through protein kinase G. J Cell Physiol., 2014; 229(12): 2142–2152. doi: 10.1002/jcp.24677. PMC4149598

Vascular endothelial cells and their actin microfilaments align in the direction of fluid shear stress (FSS) in vitro and in vivo. To determine whether cofilin, an actin severing protein, is required in this process, the levels of phospho-cofilin (serine-3) were evaluated in cells exposed to FSS. Phospho-cofilin levels decreased in the cytoplasm and increased in the nucleus during FSS exposure. This was accompanied by increased nuclear staining for activated LIMK, a cofilin kinase. Blocking stress kinases JNK and p38, known to play roles in actin realignment during FSS, decreased cofilin phosphorylation under static conditions, and JNK inhibition also resulted in decreased phospho-cofilin during FSS exposure. Inhibition of dynamic changes in cofilin phosphorylation through cofilin mutants decreased correct actin realignment. The mutants also decreased barrier integrity as did inhibition of the stress kinases. These results identify the importance of cofilin in the process of actin alignment and the requirement for actin realignment in endothelial barrier integrity during FSS. Cell Biochem. 2013; Apr; 114(4):782-95. doi: 10.1002/jcb.24416. PMC4096802

The prevalence of exercise-induced bronchoconstriction is reported to be high among recreational and elite athletes, yet diagnosis is often symptom-based. Indirect challenges such as the laboratory exercise challenge provide objective criteria for proper diagnosis and treatment. However, a standardized protocol using appropriate exercise intensity, duration, and dry air inhalation is often not implemented, and thus a false-negative test may result. This article reviews and describes the symptom-based diagnosis, the exercise challenge, and other indirect challenges such as eucapnic voluntary hyperpnea, hypertonic saline inhalation, and inhaled powdered mannitol as methods to diagnose and evaluate exercise-induced bronchoconstriction. Advantages and disadvantages of each diagnostic procedure are presented. J Allergy Clin Immunol. 2008;122:238-46.

This study was designed to investigate PM(1) inhalation during exercise on lung function, exhaled nitric oxide (eNO), and total nitrate (NO3), S-nitrosoglutathione (GSNO), and malondialdehyde (MDA) in exhaled breath condensate (EBC). Inhalation of combustion-derived PM is associated with adverse respiratory health. A mechanistic action of PM on lung function is not defined; however, nitrosative/oxidative stress is likely. Prior to and after two 30-min exercise bouts 4-5 days apart, inhaling low (7382 +/- 1727 particles cm(- 3)) or high (252,290 +/- 77,529 particles cm(- 3)) PM(1), 12 nonasthmatic males performed spirometry and eNO and EBC collection. Normal resting lung function did not change after low PM(1) exercise. After high PM(1) exercise, FEV(1) and FEF(25-75) fell significantly (p = .0005, p = .002) and was related to [PM(1)] (r = -.55, p = .005 and r =-.61, p = .002; respectively); 11- and 52-ml decreases were calculated for each 20,000 particles cm(- 3) increase for FEV and FEF(25-75). NO3 did not change after low PM(1) exercise (30.5% increase), but significantly decreased by 43.8% after high PM(1) exercise, and correlated with lung function changes (r = .63, and r = .54 for FEV(1) and FEF(25-75), respectively; p = .001 and p = .007). No change in GSNO was observed. Alveolar NO decreased after high PM(1) conditions (p = .02); eNO pre-to-post difference was related to changes in FEV(1) (r = .60, p = .002). MDA increased 40% after low PM exercise (NS) and increased 208% after high PM exercise (p = .06). Thus, high PM(1) inhalation during exercise caused a reduced alveolar contribution to eNO; NO3 and eNO variables were decreased and were related to impaired lung function. Decreased NO(3) and eNO may be due to superoxide/NO formation of peroxynitrite, resulting in lipid peroxidation. Inhal Toxicol. 2008 Jan;20(1):1-9.