Infective endocarditis (IE) is a life-threatening bacterial infection of heart valves. In comparison to treating other diseases, IE treatment is especially difficult to treat due to an increased resistance to conventional antimicrobial agents as well as a continuously high shear environment of the endocardium wall. Even after receiving prompt therapy, patients with streptococcal endocarditis often develop complications- including heart failure, progressive valve destruction, and stokes. Though the interactions between adhesive bacteria such as streptococci group of viridians (the major cause of IE) and platelets are known to facilitate bacterial vegetation in damaged heart valve, it is not well known how the bacteria bind through hitchhiker binding events (a specific interaction of platelets with streptococcus group viridans). Mainly, it is unclear whether bacteria bind to platelets and then to heart valves, or if the bacteria bind to platelets which are already found on inflamed heart valves.  To successfully characterize these spatial and temporal events, we used a parallel microfluidic device to develop a model. Specifically, we used our humanized in vitro model of infective endocarditis to understand the initial stages of the disease. We are demonstrating that the number of adherent bacteria can be measured after bacteria mixed with various blood components is washed through to the device, with sufficient accuracy to test hypotheses regarding the adhesion phase. Finally, we quantified the number of bacteria in vegetation after a growth phase following the adhesion phase. The findings from this design can potentially lead for the development of novel molecular therapeutic mechanisms. These will have implications not only for infective endocarditis, but also all other endovascular infections with a shear environment.

Spinal cord injury (SCI) currently affects approximately 285,000 people in the U.S. with more than half experiencing incomplete tetraplegia. Currently, there are no effective treatments, leaving patients with devastating lifelong ailments. This project investigates a new implant to aid treatment and recovery after SCI by mimicking the mechanical properties of the native spinal cord tissues and improving neuro-regeneration through modulation of macrophage phenotypes. Results previously produced in the Ratner Lab for engineered biomaterials research show that scaffolds with a specific porous structure create pro-healing and anti-scarring effects. Based on these results, we hypothesize that when the porous structure is paired with suitable mechanical properties, the macrophage response will be strongly indicative of a pro-healing environment. Proper mechanical properties have been achieved through variation in the chemical composition, water content, and methods of synthesizing the scaffold to preserve the microstructure. Now, the stiffness is tunable over several orders of magnitude and has been matched to the softness of spinal tissue. The scaffold also demonstrates very good biocompatibility, verified through cytotoxicity and endotoxin tests, as well as in literature. To test how cells interact with the implant, I seeded macrophages into the 3D structure and observed the response. If the cells integrate healthily into the softer 3D scaffold, this implies that the material may be a useful tool in treating SCI. To further test this, our collaborators in the Hofstetter Lab will implant the material in rats to investigate the influence on SCI recovery.

QRS-like artifacts are present in ECG recordings during CPR but have not been described previously. We coin these artifacts compression induced complexes (CICs). They prevent accurate real-time cardiac rhythm characterization during CPR compressions. Additionally, we hypothesize that CICs may be an early prognostic tool for return of rhythm (ROR), return of spontaneous circulation (ROSC) and an indicator of cardiac health. The objective is to characterize and describe these QRS-like complexes quantitatively and create an algorithm that distinguishes CICs from native cardiac activity. CIC characteristics that are being measured are a) amplitude, b) sharpness, and c) temporal relationship with CPR compressions. Custom software was used to analyze the ECG data from King County Emergency Services with CICs annotated by expert review. 50 cases were used to determine the range of features specific to CICs, to include: a) amplitude as measured at CIC peaks, b) sharpness as quantified by measuring the peak of the second derivate at CIC locations and wavelet transform analysis to measure the range of frequencies present in CICs, c) the temporal relationship between CICs and CPR compressions as measured using peak finding algorithms and comparing the delay between the impedance peaks from CPR compressions and CIC peaks. Ten CICs from each case for each feature were analyzed using standard deviations to define quantitative parameters for a CIC detection algorithm. After development, this algorithm was tested to determine the percent of CIC complexes that are detected in a test set. It was also used to examine the association of CICs with ROR and ROSC. This algorithm can potentially aid in advising pre-hospital care tailored to the patient’s cardiac condition.

Epigenetic modifications regulate chromatin structure and function, playing important roles in altering DNA transcription levels and subsequently cell fate decisions. Various next-generation sequencing (NGS) methods have been developed to detect these epigenetic changes in the genome, such as chromatin immunoprecipitation followed by sequencing (ChIP-seq). Even though ChIP-seq is extensively used to analyze DNA and histone modification levels, this method is limited to one histone marker at a time and requires significant amount of input cells, which masks the profiles of cell-to-cell variation and the complex interaction between the epigenome and gene expression. To overcome these limitations in current next-generation sequencing methods, we have been developing a multiplexed assay that could detect multiple epigenetic modifications in single cells. So far, I have developed a NGS data analysis pipeline to identify potential gene candidates that are highly differentially modified by histone markers. In the future, we hope to use these gene candidates as templates to design DNA-fluorescent in situ hybridization (DNA-FISH) probes and perform Expansion Microscopy and DNA-FISH with these probes to link histone modifications to specific gene loci at high resolution. After the assay is fully developed and validated, we plan to utilize the assay to take the epigenetic profiles of hematopoietic stem cells and study cell fate decisions in hematopoiesis.

The misfolding and aggregation of free light-chains into amyloid fibrils is the hallmark of antibody light-chain (AL) amyloidosis, a fatal disease associated with the accumulation of amyloid species in tissues throughout the body, including the heart and kidneys. Current treatment options, including chemotherapy and bone marrow transplant, do not address the causes of aggregation on a molecular level. Molecular dynamics (MD) simulations were used to investigate misfolding pathways in the aggregation of two light chain monomers, Jto and Wil. These simulations showed that under amyloidogenic conditions, conversion from beta-sheet to alpha-sheet secondary structure was observed in both Jto and Wil. Misfolded conformations, obtained from the MD simulations, were used to guide the design of alpha-sheet peptides, which have been used previously to inhibit amyloid formation in diverse systems. The designed peptides were evaluated computationally by docking them against misfolded conformations of Wil, and the best performing peptide was chosen for future experimental work to explore its potential to limit aggregation.

Type 2 diabetes (T2D) is a disease associated with pancreatic islet β-cell failure; especially, the loss of β-cell mass. Approximately 75% of patients who start with one medication will need multiple to control the progression of this disease. With no cure and numerous secondary complications - blindness, kidney failure, heart attack, and stroke - T2D places an enormous burden on our society and healthcare system today; The disease is projected to be the seventh leading cause of death by 2030. Islet amyloid polypeptide (IAPP) deposition is observed in approximately 90% of T2D patients. While human IAPP (hIAPP) is amyloidogenic, forming amyloid fibrils and deposits, IAPP from rodents is not. Our group’s previous findings suggest that T2D and other amyloid diseases form soluble toxic oligomers through a non-standard α-sheet secondary structure. We propose a peptide-based therapeutic to combat T2D via utilizing synthetic α-sheet compounds complementary to the amyloid-associated α-sheet structure to target the toxic oligomer form of hIAPP. In this study, we addressed the efficacy of this peptide-based therapeutic through inhibiting the aggregation of hIAPP via a Thioflavin-T assay and neutralizing the hIAPP oligomer cytotoxicity via a MTT cell viability assay in a human-derived pancreatic cell line. The preliminary results suggest that our peptides are effective in reducing hIAPP aggregation and oligomer cytotoxicity. Moving to a more biologically relevant model, we evaluated the effects of our compounds on reducing amyloid formation in the islet of Langerhans from transgenic mice; the preliminary results are encouraging. The α-sheet platform provides a novel potential therapeutic for treating T2D and other amyloid diseases.

Alzheimer's disease affects millions of individuals worldwide, yet there is an astounding lack of marketed treatments that can effectively slow the neurodegeneration associated with the disease. In the past few decades, evidence has emerged that small, soluble aggregates of beta-amyloid (Aβ) peptide known as oligomers are primarily responsible for toxicity in the brain, which has sparked research to develop inhibitors targeting the toxic oligomers. However, the actual structure of Aβ oligomers remains unknown, in large part because traditional methods used to determine protein structure are ineffective due to the dynamic and heterogeneous nature of these oligomers. This in turn has greatly hindered therapeutic development. Interestingly, a designed α-sheet peptide known as AP3 displays striking behavioral similarities to Aβ under low pH, making it an ideal model for understanding amyloid protein behavior. In addition, AP3 aggregation and toxicity are potentily inhibited by other amyloid species. The interactions between this synthetic amyloid and naturally-occurring amyloid species, including Aβ, are being explored both experimentally and via computer simulation, and the data are being used to design a de novo peptide inhibitor. This inhibitor is being tested for its ability to inhibit amyloid aggregation and toxicity, as well as whether it can specifically bind to heterogeneous populations of Aβ even at low concentration. The successful completion of this project wil result in significant progress towards understanding amyloid behavior and aggregation. This may eventually lead to novel applications of the α-sheet structure in treatments and diagnostic assays for Alzheimer's.

Streptococcus mutans is an acidogenic bacterial species that predominates in the oral microbiome. S. mutans binds to the salivary pellicle, a layer of proteins that forms on the tooth surface and forms acids after metabolizing sugars. Accumulation of S. mutans biofilms leads to cavity formation, and when the bacteria travels to and infects the heart, can lead to infectious endocarditis. Therefore, decreasing accumulation of S. mutans is a key concern. Recent evidence suggests that S. mutans is among the bacterial species that utilize functional amyloid fibrils in its biofilms. Amyloids are insoluble fibrillar protein aggregates with a cross-ß structure, and functional amyloids are used by bacteria to provide structure and strength to their biofilms. While functional amyloid systems in bacteria such as E. coli and S. aureus have been investigated, very little is known about the mechanism or purpose of S. mutans functional amyloids. Polyphenolic small molecule epigallocatechin gallate, or EGCG, is an amyloid inhibitor in S. mutans biofilms. Previous results from our lab suggest that amyloid fibrillization progresses via an intermediate that adopts a unique secondary structure, an alpha-sheet. Alternating L- and D- amino acid peptides adopt an alpha-sheet secondary structure and have been shown to inhibit amyloid formation in multiple mammalian systems by binding to soluble alpha-sheet-containing oligomeric species. Inhibition of amyloid formation by alpha-sheet peptides suggests presence of an alpha-sheet intermediate species on the pathway to functional amyloid formation. To investigate the mechanism of functional amyloid formation in S. mutans, alpha-sheet peptide inhibitors were compared to EGCG for their ability to inhibit fibril formation of S. mutans adhered to an artificial salivary pellicle.

As one of the five leading causes of blindness, uveitis is the reoccurring inflammation of the uvea, demonstrating pathological changes in the eye. In recent years, optical coherence tomography (OCT) has been recognized as the leading, clinically accepted imaging modality for diagnosing major human optical diseases. Despite this, research on the clinical usage of OCT, primarily spectral domain OCT (SD-OCT) and swept source OCT (SS-OCT), for uveitis diagnosis remains sparse due to the lack of a quantitative-based set of parameters to assist with OCT image analysis. As a result, there is a need to develop an index of parameters that quantifies the microvasculature and structural changes associated with uveitis. To address this need, a novel five parameter quantitative-based metric consisting of distance of retinal detachment, retinal thickness, vessel area density, vessel diameter, and vessel perimeter was evaluated. Through layer segmentation of SD-OCT and SS-OCT scans, application of optical microangiography, and quantitative analysis of structural and microvasculature changes for healthy and uveitis cases, the clinical potential of SD-OCT and SS-OCT for diagnosing uveitis was evaluated. This project introduced a metric for evaluating changes associated with uveitis in a qualitative and quantitative manner to further understand the abnormalities that accompany the disease. Assessing the clinical efficacy of SD-OCT and SS-OCT for uveitis detection can provide insights on the most effective method for diagnosing this disease.

DNA origami nanotechnology has evolved rapidly since its conception eleven years ago. Both two-dimensional and three-dimensional nanostructures have been created with potential applications in targeted drug delivery, “smart” diagnostic technology, and the study of cell behavior. By annealing “staple” oligonucleotide strands to a single-stranded DNA scaffold we can effectively fold the DNA onto itself to build the nanostructures of interest. One of the primary physical limitations to what one can build is the scaffold. The most commonly used scaffold is derived from the bacteriophage M13mp18 and has a length of 7,249 nucleotides. Its length has previously been varied; however, an overlooked limitation is the secondary structure DNA naturally exhibits. These are sites in which the scaffold binds to itself, thus creating competition for staples to bind during folding reactions. To predict the impact that a designed sequence with little secondary structure could have, we analyzed the first 6,000 bases of the M13mp18 DNA sequence using NUPACK, a nucleic acid sequence analyzer, for their minimum free energy (MFE) at storage, manipulation, and maximum folding reaction temperature. Preliminary data shows M13mp18 exhibits less secondary structure at a high temperature (65°C) than at a low temperature (4°C) and increasing the concentration of divalent salts linearly increases the amount of secondary structure. Additionally, alternative, shorter sequences have been engineered and their secondary structure is being analyzed at varying conditions. To further determine the effects on yield and stability, structures will be folded using the designed sequence and the standard sequence as a scaffold. These will be compared through agarose gel electrophoresis and transmission electron microscopy. The results from this preliminary data could help us move us toward using a scaffold with decreased secondary structure present at folding temperatures which could potentially result in higher yields, shorter folding reactions, and increased stability.

Characterizing biological functions on a single molecule scale increases understanding of biological functions by providing information about the indiviual contributions which combine to create larger scale functions. Single molecule measurements are a crucial part of characterizing molecular interactions. Although atomic force microscopy (AFM) and magnetic tweezers are able to measure the response of single molecules to mechanical force, it is challenging to ensure single molecules are being measured. In this project, a precise DNA Origami structure was used to space molecules for single molecule force measurements. Base pair association between DNA nucleotides allowed specific nanostructures to be designed and fabricated. Molecules of interest self-assemble to specific sites of the structure. AFM was used for imaging and obtaining force measurements. This research investigates the strength of adhesion for double stranded DNA when subjected to different loading rates as a proof of concept. In the future, this structure will be used to determine force properties of diverse molecular interactions like platelet and bacterial adhesions.

Tyrosine kinases are critical drug targets in oncology due to their role in tumorigenesis. Accordingly, the treatment of specific cancers has been revolutionized by the development of tyrosine kinase inhibitors (TKIs). However, the long-term effectiveness of TKIs is undermined by the emergence of drug resistance, often caused by mutations that prevent drug binding. Understanding whether a given mutation confers drug resistance can enable physicians to tailor treatment based on the patient’s tumor genotype. Current methods to identify resistance mutations are laborious and can only interrogate a small subset of possible mutations. To overcome this limitation, I have designed an assay to identify all possible single drug resistance mutations in the oncogenic kinase ALK in a single experiment. A key feature of this functional assay is that it can accurately discriminate between drug resistant and drug sensitive mutations. Moreover, this assay leverages the Ba/F3 cell line, a mammalian cell line that only proliferates when an active oncogenic tyrosine kinase variant is expressed. Thus, when treated with a TKI, cells expressing drug resistant kinases continue to proliferate while cells expressing sensitive kinases die. As a first step, I have genetically engineered the Ba/F3 cell line to enable single-copy integration of tens of thousands of ALK variants. Additionally, I have cloned ALK into a mammalian expression vector and have optimized transfection conditions for the Ba/F3 cell line. Next, I will transfect the cloned vectors into Ba/F3 cells and measure the growth rates of each transfected cell line. I anticipate that only cells harboring active ALK will grow, while those with inactive variants will not. This result would lay the foundation for further development of a system for the exhaustive identification of drug resistance mutations in oncogenic kinases.

Paper fluidic devices expanded diagnostic medicine to applications beyond lab testing. A key example is the common pregnancy test, which relies on an antibody capture line and detection antibodies conjugated to a colorimetric label to provide a diagnostic readout. This detection method requires high concentrations of target proteins. However, by incorporating isothermal strand displacement amplification (iSDA) and antibody capture of proteins tagged with nucleic acids (rather than colorimetric labels) much smaller protein concentrations can be detected. The Yager Lab has shown that this method increases sensitivity by 10⁴ in comparison with conventional lateral flow methods. Incorporating iSDA into a multistep fluidic device poses the challenge of transporting, delivering, and holding solutions with accurate concentrations of iSDA reagents in an amplification region for thirty minutes. A specific difficulty is delivering the flow of rehydrated iSDA reagents and subsequently stopping fluid movement over the protein capture region. My initial work has been focused on designing and optimizing a device with stopped flow to create ideal amplification conditions evaluated with fluidic dye testing. By using fluorescein and ImageJ analysis to iterate device and membrane design, correct concentrations were delivered to the amplification region. To further the device automation, timers were incorporated so the only user step would be inputting the sample. The device then isolated the target protein, amplified the signal, and provided a diagnostic analysis for the user using a fluorescent probe. Such an advancement can further the reach of higher sensitivity protein diagnostic technologies to low resource settings and for in home testing.

Synthetic biology allows us to use an organism’s metabolic pathways to produce new, useful metabolites cost-effectively. Growing organisms in a culture allows the production of these metabolites on a commercially-viable scale. However, management of these cultures is time-consuming and labor-intensive, such as HPLC. Our project aims to provide a low-cost, automated system that analyzes the culture in real time at low cost. Our device, a turbidostat bioreactor called the Chromastat, uses images and other optical data to measure the current state of a culture. If these measurements show metabolite production outside specifications, the system modifies the culture’s metabolic expression by adding inducers. To make this system cost effective, we focused on using affordable materials, an open-source controller and analysis system, and visible color outputs instead of the traditional fluorescence. To test our system, we used the violacein metabolic pathway in yeast. By regulating gene expression with inducible promoters, this pathway yields up to four visually distinct pigments. In a production environment, these pigments could provide a proxy measurement to produce useful substances which are costlier to detect. Pigment production is measured by the popular low-cost and open-source Raspberry Pi computer running a program displayed in Java Swing. Optical sensors collect color and opacity information about the culture. Our software, installed on the Raspberry Pi, analyzes the color, and in response, the hardware introduces inducers to stabilize the culture’s production. Data recorded by the Pi over time reveals the relationship between gene expression and metabolite production rates. By combining biological, software, and hardware systems, our unique design can generate previously unavailable visual data in certain biosynthesis processes, such as those involved in antibiotic production or fermentation.

Traumatic spinal cord injuries may lead to devastating loss of neurological function and there are currently no effective clinical methods to regenerate damaged nerve tissues. Besides, the inflammatory response can cause secondary injuries and inhibit regeneration. Novel treatments like cell therapy and localized drug delivery have shown greater efficacy when delivered by hydrogel scaffolds. However, effective scaffolds that can both culture neuronal cells and deliver therapeutic drugs are rare. Therefore, there is a need for an effective treatment of spinal cord injury that can locally deliver therapies with anti inflammatory properties to the spinal cord, as well as provide a support material for neural stem cell therapy. Here, we report a hydrogel composed of peptides that can self-assemble into a hydrogel in physiological conditions. The self-assembling peptide is conjugated to a polymer that enables drug loading and bioactive properties. Self-assembling peptides and a drug loading polymer were synthesized and characterized, loaded with bivalirudin, a model anti-inflammatory peptide drug, and assembled into a hydrogel formulation. The hydrogel was used to encapsulate neural stem cells to assay its efficacy as a support material. The gel was also assayed for its ability to release the anti-inflammatory drug when exposed to enzymes upregulated during inflammation. We anticipate seeing effective ligation of the polymer with the self-assembling peptide, successful hydrogel formulation, and neural stem cell viability when encapsulated in the hydrogel. A successful self-assembling formulation will ultimately work to provide effective cell therapy and anti-inflammatory drug delivery to the spinal cord, with potential for broader drug loading applications in the future.

Antiretroviral therapy (ART) is the standard of care for treating human immunodeficiency virus (HIV) infection and suppresses virus levels but cannot eliminate latent HIV reservoirs. Latently infected cells are a barrier to HIV cure. “Shock and kill” is a strategy that uses latency-reversing agents (LRAs) to reactivate latently infected cells under suppressive ART, making the cells vulnerable for removal by host immune responses or other strategies. However, “shock and kill” fails in clinical studies due to the low potency and high toxicity of LRAs. Here we have developed a lipid-polymer nanoparticle platform that addresses “shock and kill” limitations by incorporating multiple LRAs to increase potency and to reduce toxicity by targeting HIV reservoirs in lymphatic tissues. We have shown that ~200nm nanoparticles sustain LRA release and induce latency reversal in a human T-cell line in vitro. To target lymph nodes, we have synthesized nanoparticles smaller than 100nm in diameter, which literature shows enhances lymph node targeting following subcutaneous injection. By adjusting the polymer and lipid concentrations as well as the organic to aqueous solvent ratio during formulation, we obtained an ultrasmall particle of ~100nm. Ex vivo organ imaging from mice subcutaneously administered these ultrasmall particles shows successful targeting to draining lymph nodes as well as other HIV reservoir locations, including the spleen and the gut-associated lymphatic tissue. The ultrasmall nanoparticles also show similar drug release and latency reversal properties as our previous formulations. The nanoparticle platform demonstrated here is able to target latent HIV reservoirs in multiple areas of the body, which is crucial for eliminating latent HIV reservoirs and achieving a cure.

Recent advances in RNA sequencing and molecular biology has allowed them to be at the forefront of diagnostic care. Although sequencing costs have decreased, existing methods for detecting gene expression levels are not feasible in a clinical setting. Creation of a simpler molecular diagnostic based on measuring gene expression levels in blood could provide an alternative as a reliable, fast and easy to perform diagnostic. Multiple genes can have various expression levels across patients and patterns of this differential gene expression is key in studying multiple diseases including cancer or autoimmune disorders. Before testing the platform with clinical samples a smaller classifying circuit that fluorescently differentiates between a series of different cancer cell lines was created. Directly, my work has been developing fluorescent reporter probes that will be able to categorize weighted genes in our classifiers neural network. Positive and negative gene weights can be used to differentiate between cancer cells lines as cells express certain genes in varying amounts between populations. When running samples in our qPCR protocol, addition of the fluorescent probes, allows each gene to be sorted into distinct fluorescent channels. After testing the probes using RNA extracted from our cancer cells (HEK, HEPG2, MCF7) results were similar to the known gene expression data when measuring a series of 5 genes across these cell lines. Additional, experiments are currently underway to establish a gradient for fluorescence so that not only the type of cancer cell can be determined, but the proportional amount with high specificity and low variability. After development the fluorescent probe system will be incorporated into a clinical sample protocol and the platform will undergo further optimization for clinical applications. For patients a novel diagnostic platform allows for a more thorough disease screening, longer treatment time and hopefully a more positive prognosis outcome.

Every year there are at least one million new cases of sport-related concussion in children younger than 18 in the United States. Current diagnostic screening tools, such as CT and MRI, are insensitive to the subtle microstructural changes that occur following concussion, and in pediatric concussion, there is additional complexity of the still-developing brain and how concussion affects its long-term development. While these patients’ radiographic images often appear normal, they report an array of post-injury symptoms, which questions the true extent of brain injury after concussion. The aim of this research project is to utilize advanced neuroimaging and analysis tools, diffusion tensor imaging (DTI) and graph theory, to explore short-term and longitudinal changes in the brain following pediatric sports concussion and to obtain a more reliable and sensitive method to diagnose pediatric concussion. Children aged 10 – 14 with unresolved symptoms from a sports-related concussion and age-matched controls were included in this study. Each participant underwent MRI scans and clinical assessments 4-6 weeks post-injury and 6 months after the initial visit . We used DTI, which has been shown to be sensitive to microstructural changes related to concussion in adults. In addition, we analyzed the DTI data using graph theory, which is a mathematical tool that models information as a network. We investigated differences in network properties between concussed and non-concussed children and used random generated networks as a control. We confirmed that the network properties of children were distinct from random networks. We also observed a 9% reduction in global clustering and 16% increase in local connectivity in the concussed patients, suggesting overall network disconnect and stronger, but more segmented, local network groups. These preliminary results encourage further exploration of the methods employed and display clinical relevance in distinguishing between concussed and non-concussed youth. 

Zika virus has become a major epidemic around the world due to the serious birth defects that it can cause; this has led to an increase in research regarding Zika and how to diagnose the disease. Diagnostic tests require a specific sample type to be tested for the presence of a virus, and because it has been shown that Zika viral levels are more consistent and reliable in whole blood compared to plasma or serum, whole blood is the more efficient sample type. Current strategies for diagnosing Zika fall short because there is limited data on how the virus presents itself in the body, and many of these strategies utilize expensive and time-consuming methods that are not feasible in low-resource settings where Zika is more prevalent. Therefore, there is a need for a low-cost and instrument-free process that can effectively purify Zika virus RNA from whole blood in order to allow for detection of the viral RNA. This project dealt with optimization of an existing lab protocol that required laboratory equipment and resulted in low efficiency. The aim was to develop a robust, high-efficiency, instrument-free RNA purification protocol for whole blood samples. The method of RNA purification consisted of a lysis and binding buffer, followed by RNA purification using silica beads embedded in paper to extract pure RNA from the sample. qRT-PCR was then used to quantify the resulting RNA. So far, the paper-based extraction method recovers approximately 20% of the initial target RNA; however, there are still some techniques that will be employed in the future to help improve this recovery rate. The goal of this project is to increase RNA purification efficiency and to provide an alternative mechanism to purify nucleic acids that can be used in a Zika virus diagnostic test.

At the 2017 Undergraduate Research Symposium, I presented a low-cost, automated device for influenza detection that was being developed in the Yager lab. After creating, testing, and optimizing this device, we were left with many questions. While certain components of fluid flow in paper are understood, there is much more to learn. In this device alone, three different types of paper were used to run the two-dimensional assay. Current unanswered questions include the following: how do additives affect protein deposition, rehydration, and capture? How much of a dried protein on paper can be rehydrated by a passing solution, and which paper and solution properties affect this value? How do the viscosity and complexity of our patient samples affect the quality of our results? Our overall goal is to determine how to optimize our test line and generate a better understanding for optimization of future diagnostic devices. To make progress towards this goal, my project centers on the development and characterization of a laboratory technique to quantitatively study the effects of these parameters on protein interactions with membrane surfaces and other proteins bound to those membrane surfaces. Improving our understanding of these interactions in membranes will enable better diagnostic device optimization and enable illness detection with lower amounts of infected sample – allowing for earlier detection and better disease treatment. Through the development of this testing apparatus, I have begun to address these questions.

Cancer is the 2nd leading cause of death in the United States. Of these cases, 6-7% are pancreatic cancer (PC), yet this disease is ranked 1st in mortality because what causes PC and how it manifests is still relatively unknown. Early detection is difficult and in a majority of cases, patients are diagnosed after the cancer has progressed to final stages. For the last 7 years, the Human Photonics Laboratory (HPL) has been dedicated to early detection and diagnosis of PC through the next generation 3D pathology (and all aspects of its infrastructure: 3D microscopy, 3D reconstructions/supercomputing, millifluidic device development/whole tissue processing, clinical evalution/validation). For PC, cell and tissue specimens which have been imaged in 3D and reconstructed in an advanced 3D visualization software (Amira) require an appropriate gray scale and RGB color scheme to determine accurate cancer diagnoses. Ideal and non-ideal samples may then be passed onto collaborating pathologists for clinical evaluation and validation. This research project seeks to satisfy three primary aims: (1) properly match the color metrics/scheme of the raw optical 3D microscope datasets to 3D computer reconstructions and analyze (qualitatively/quantitatively) the gray scale/RGB color histogram between normal/cancerous (human) pancreatic samples, (2) compare/contrast the pathological metric known as the nuclear-to-cytoplasmic (N/C) ratio between a small sample of normal/cancerous specimens, (3) choose 5-10 ideally processed/matched specimens and submit to pathologists for clinical evalution. The outcome of this project is expected to accelerate the diagnosis of PC.

Lateral flow tests are assays conducted on porous media, commonly referred to as paper, that can detect and quantify analytes in a sample. They have the potential to return results within minutes and can be used in low-resource or non-medical settings. Protein adsorption is critical in these tests because protein interaction with a media can affect the delivery of reagents or choice of blocking agents to prevent nonspecific binding. A computational model of the fluid and adsorption physics may improve the development of paper-based assays. Yet-to-be-published data from the Yager group evaluates protein adsorption based on the deposition and analysis of protein spots on paper. The computational model used is derived from Darcy’s law, commonly used to model fluid flow in porous media and to calculate volumetric flow rates. This model makes two main assumptions: the porous media is fully saturated, and there is zero outflow on the boundaries of the media. These assumptions are not applicable because the protein spot dries as a result of evaporation, creating a non-zero outflow at the surface of the paper. The saturation level in the paper will also vary therefore Darcy’s law is not an adequate representation of the paper. My project aims to develop a more accurate computational model of fluid and protein absorption dynamic by modeling protein spots on paper. I incorporated an evaporation component that will allow the use of Richards equation to model fluid flow, which applies to unsaturated conditions. This is coupled with a protein transport and adsorption component to achieve a complete model. Although literature on evaporation from sand or soil is abundant, these results cannot be directly applied to evaporation from paper. This model will aid the diagnostic community in better understanding their assays and ultimately help improve the sensitivity and specificity of their assays.

Regenerative medicine that utilizes pluripotent stem cells (PSCs) has the potential to transform the treatment of the many debilitating conditions that face modern society. Although research regarding the control and use of PSCs is prevalent, there has been little translation of this research to the clinic. Critically, there remain significant knowledge gaps regarding peri-gastrulation cell fate decisions — the decisions at an early phase in development during which pluripotent stem cells first begin to differentiate toward specific cell fates. Currently, the widely used systems for understanding cell fate decisions rely on differentiating populations of PSCs toward a single target cell lineage in a well plate via uniformly applied morphogen signals. Such uniform stimulation lacks the ability to reproduce the same spatial cell fate decisions as is observed during embryogenesis. We have developed a technology that establishes in vitro morphogen gradients to better recapitulate peri-gastrulation cell fate decisions. This research focuses on precision cell colony patterning using CNC milled and stereolithography 3D printed constructs to better characterize the effect of in vitro morphogen gradients on single populations of PSCs. This was done by patterning PSC colonies and subsequently forming morphogen gradients of Activin A and BMP-4/CHIR99021 to establish subpopulations of ectodermal, mesodermal, and endodermal lineages. The growth factors Activin A and BMP-4 and CHIR99021 (a small molecule agonist of Wnt signaling) are biochemical factors that influence signaling pathways which control early cell fate decisions. The cells were then fixed and stained for markers of ectoderm (CDX-2), mesoderm (Brachyury), and endoderm (Sox2) to quantify the induced subpopulations. The ability to control cell fate decisions of PSCs is an integral step in developing functional and effective regenerative medicine technologies. Ultimately, this technology has the potential to develop more effective induced differentiation techniques for creating therapeutic, mature cell populations.

In the US, someone needs blood every two seconds, making blood transfusion a frequently performed procedure in hospitals. Transfusion of incompatible blood due to clerical error in clinical blood typing procedures can induce a multitude of life-threatening immune responses in the recipient’s body. The most prevalent typing procedures are based on the overly simplified characterization of surface antigens on an individual’s red blood cells (RBCs) into the commonly known ABO blood type system. However, studies have shown that the antigens embedded in the RBC membrane, comprising of glycolipids and glycoproteins, are structural determinants of variations in the blood type outside of the ABO system, elucidating the limitations of current typing methods. This project follows the hypothesis that gas-phase hydrogen/deuterium exchange (HDX) and ion mobility mass spectrometry (MS) techniques can be employed to characterize and further elaborate the structure of these RBC antigen constituents. To test this hypothesis, genotypically defined donor blood samples undergo an osmotic lysis procedure to obtain RBCs ghosts, which are RBC membranes without the cellular components. Glycoprotein and lipid fractionation, and digestion is employed to isolate carbohydrate components in preparation for MS analysis which provides insight into structural composition of the carbohydrate antigens. Preliminary analysis on the donor samples has suggested the presence of clinically relevant carbohydrate antigens.This knowledge will be crucial in further developing glycobiological sensors that can be functionalized onto silicon chips used by Ratner Lab, facilitating novel blood typing techniques. Through this study, we expect to develop a deeper understanding of the heterogeneity encoded into the carbohydrate RBC antigens which play a critical role in transfusion medicine.

The PTEN (phosphatase and tensin homolog) protein negatively regulates growth-promoting PI3K-Akt signaling in cells. Due to its function as a tumor suppressor, PTEN is often mutated in diverse cancers. Unfortunately, most PTEN variants have not been individually studied, making it difficult to ascertain their functionality within cells. Our lab recently demonstrated that thousands of PTEN missense variants exhibit decreased steady-state abundance when expressed in human cell lines and likely have reduced function. However, the mechanism behind their lower abundance is currently unknown. We hypothesized that many low-abundance PTEN variants are thermodynamically unstable and possess a reduced melting temperature. To test this, we fused EGFP, Enhanced Green Fluorescent Protein, to a panel of PTEN single amino acid variants, and these fusion proteins were expressed within human cell lines. Then, we cultured these cells at their standard growth temperature (37°C) and two lower temperatures (33°C and 30°C). We found that a subset of variants of intermediate abundance at 37°C exhibit WT-like abundance at decreased temperatures, while variants of extremely low-abundance remain unchanged. These results suggest that we can identify missense variants with reduced thermodynamic stability using this method. Next, we will repeat this experiment at high throughput to identify hundreds of temperature-dependent PTEN variants. We will determine biochemical properties shared by partially stable variants and compare them to computational predictors of protein folding. We will also identify variants of intermediate abundance that are not temperature-dependent, which may reveal other mechanisms by which variants lower a protein’s abundance. Our results demonstrate that we can characterize the thermodynamic stability of PTEN variants by measuring their abundances in cells grown at different temperatures. These results might be more physiologically relevant because the variants were studied in a cellular environment. Furthermore, our methods may be applied to other proteins that cannot be studied as purified protein.

The majority of urinary tract infections are caused by E. coli bacteria. E. coli infections are strengthened due to E. coli’s ability to bind to mannosylated cells. A receptor protein on E. coli, FimH, has two domains: a regulatory pillin domain and a lectin domain (LD). In the presence of a force, the pillin domain detaches from the LD allowing the LD to go from its low to high affinity state. The naturally occuring mannose sugars on Horseradish Peroxidase (HRP) are known to bind the LD of FimH. The mannose binding pocket is suspected to open transiently, even when bound to mannose sugars. Based on the interaction of HRP with FimH, HRP may not regularly dissociate during these episodes, but we suspect that free mannose can induce the dissociation of HRP from FimH in these moments. To determine if mannose can improve HRP dissociation from LD, we are using an assay similar to a competitive Enzyme Linked Immunosorbent Assay. We expect to see a decrease in HRP bound to LD in the presence of free mannose compared to the absence. Ultimately this experiment provides an explanation of mannose monomers as an inhibitor for longer chains of mannose binding and a premise for a larger study on alternatives to E. coli antibodies that can competitively inhibit FimH from binding mannosylated cells.

Excessive and unchecked deforestation can create a cascade of extinctions. A field-deployable DNA screening tool that can be used by non-scientists will prevent illicit timber shipments from reaching a commercial market by flagging products sourced from endangered species. The DNA screening tool transports reagents and samples using the anisotropic ratchet conveyor (ARC), a portable digital microfluidic system. The screening tool recognizes the DNA barcode using species specific primers and DNA amplification indicates the identification of a species. Isothermal amplification methods are easier to integrate into the portable DNA screening tool and there is currently no isothermal amplification method for timber DNA. The loop-mediated isothermal amplification (LAMP) method was designed for integration in a portable identification system since this method has the ability to perform rapid amplification and is robust to inhibitors. The species for which amplification was performed were eastern white pine (Pinus strobus), Spanish cedar (Cedrela odorata), and white oak (Quercus alba). The first step was to identify the genetic target for amplification and design LAMP primers for this sequence. The genes selected were rbcL and matK since these chloroplast genes diverge between species and are conserved within tree species. LAMP uses six primers: the backward inner primer(BIP), forward inner primer(FIP), the forward loop primer(FL), backward loop primer(BL), forward primer(FP), and backward primer(BP). Species specific LAMP primers were designed. The primers were validated on synthetic oligos. Extraction of DNA is challenging because the DNA is trapped in between thick cell walls. Timber DNA amplification is challenging because inhibitors, include polysaccharides and phenols, bind or degrade DNA polymerase, and prevent amplification. Extraction and purification methods were investigated to provide amplifiable DNA. Isothermal amplification was performed on the ARCs. The portable DNA identification system will identify timber species for conservation biology.

T cell-based immunotherapy has shown immense therapeutic potential for the late-stage treatment of cancer. The synthetic ex vivo activation of T cells is a key step in manufacturing these therapies, inducing proliferation and cytotoxic functionalization of the cells before reinfusion into the patient. This is generally done with the use of artificial antigen presenting cells (aAPCs) consisting of antibodies triggering the receptor signaling necessary for activation directly conjugated to a spherical polystyrene bead. Although intended to mimic the function of antigen presenting cells (APCs) in the body, currently used aAPCs do not fully recapitulate natural size, morphology, or membrane fluidity, all of which have been demonstrated to be important determinants of activation properties. Therefore, there is a need for an activation platform that more closely mimics APCs in vivo, which could significantly improve the efficiency of T cell activation for use in cancer immunotherapies. To achieve this, we utilize cell-molded silica microparticles that retain the size and morphology of their cellular templates. The fusion of an antibody-loaded lipid bilayer to these silica microparticles results in a closer recreation of natural APCs. We have fabricated these cell-molded silica aAPCs with various cell template morphologies and lipid compositions. Using a variety of cell-based assays, we are characterizing the capacity of these aAPCs to induce T cell proliferation, cytokine release, and surface marker expression, all of which are metrics indicative of activation. If these results indicate that this platform successfully improves the efficiency of T cell activation as compared to current alternatives, this has the potential to improve the cost and scalability of these promising cancer treatments.

T cells of the immune system protect humans from most threats because they can recognize and eliminate foreign targets, as well as protect from reinfections. However, the persistence of an infection in the body leads to chronic stimulation of T cells, causing them to lose their effector function and enter a state known as “exhaustion”. Exhausted T cells are defined by increased expression of inhibitory receptors, loss of immune cell regulation, and most importantly, loss of cytotoxicity and effector function. Studies have shown that the transcription factors, T-bet and Eomes, play crucial roles in regulating T cell differentiation, with T-bet being highly associated with effector T cell differentiation. T-bet expression is dampened in exhausted T cells, and therefore, I hypothesize that controlled induction of T-bet expression can reverse the exhausted phenotype in antigen-experienced T cells. I constructed a T-bet overexpression vector containing T-bet cDNA fused to a fluorescent protein and destabilizing domain. The destabilizing domain, or degron, facilitates degradation of the constitutively expressed T-bet transgene in the absence of the ligand, Shield-1, which when added at varying concentrations allows for a range of protein stability. This method of overexpression confers faster control kinetics compared to commonly used transcriptional approaches, such as inducible promoters. I have characterized the range of the T-bet transgene in Jurkat cells by titrating Shield-1 to provide a working range of 5-60% overexpression of T-bet compared to endogenous levels. Validating these parameters in primary T cells will allow me to apply this T-bet overexpression vector in a mouse model of T cell exhaustion. This tool has significant implications for improving immunotherapy strategies, such as TIL and CAR-T therapies, where exhaustion of the therapeutic T cells has led to reduced efficacy of the treatment.

Current drug testing methods are unable to accurately predict and study the effects of drugs on human myocardial tissue. These methods are unable to accurately mimic the physiology and maturity of native cardiac tissue, and as a result, the tissues are unable to react to cardiotoxic drugs the same way that native cardiac tissue. Therefore, there is a need for physiologically accurate and mature tissue constructs in order to test clinical drugs for effectiveness, cardiotoxicity, and arrhythmic effects. To fulfill this need, we will use an electroconductive bioink containing decellularized extracellular matrix (dECM) from porcine cardiac tissue, reduced graphene oxide (rGO), and incorporated human induced-pluripotent stem cell (hiPSC)-derived cardiomyocytes to develop a more physiologically accurate cardiac tissue construct. This will result in more adequate cell maturation, and therefore, more accurate preclinical drug screening. The success of this project would lead to the development of high throughput production of biomimetic cardiac tissue models for use in accurate drug screening, along with improving the understanding of cardiac maturation.

Modern disease research utilizes two-dimensional (2D) stem-cell tissue culture models, simplified 3D engineered tissues, and animal organisms to study microenvironments and human physiology. However, in vitro platforms often oversimplify the physical tissue niche and animal models do not always accurately represent the function of human tissues. For instance, 2D tissue platforms used to study cardiac biology are limited because they cannot accurately recapitulate the pumping motion of the human heart which is responsible for the circulation or blood. Furthermore, animal models have an under-representative cardiovascular physiology making them inadequate systems for studying human cardiac biometrics. As such, we propose to develop a 3D tissue culture system that can accurately mimic the hierarchical organization of different human tissues. Using our novel flexible cell-sheet stacking technique, we can precisely stack layers of organized cell-sheets to create 3D laminar tissues. Then, these organized laminar tissues can be manipulated by the flexible scaffold into complex 3D tissue shapes using custom-made molds. For example, the human heart is helically aligned cardiac tissue throughout its structure that allows for the generation of intraluminal pressure during ventricular contraction. Inspired by the intricate architecture of human myocardium, this project aims to analyze how varying degrees of cell orientation can influence intraluminal pressure generating function. To account for the hollow and conical nature of the human heart architecture, we have designed a model for casting closed 3D hydrogel scaffolds with a hollow lumen that allows for intraluminal pressure measurements. The proposed model will allow for the determination of the optimal angle of cell alignment that produces the greatest intraluminal contraction. We will validate this platform using a contractile mouse skeletal muscle cell type. This approach can be adapted to model the organization of any contractile muscle tissue and be used to study the function and microstructure of human tissues.

Peripheral neuropathies involve the destruction of peripheral nerves, which leads to impairment of the neuromuscular junction (NMJ). This is the area where the nerve transmits information to muscle. NMJ breakdown will cause this synapse to degrade, generating symptoms such as reduced muscle contraction, respiration, and movement. The problem is that current efforts to develop treatments against peripheral neuropathies are severely dampened by having a lack of human-based models of synapses. Therefore, there is a need for a non-invasive method to explore and characterize synaptic function for future treatments. This research study offers a novel system to investigate the differences in neuromuscular junctions between healthy and disease states by utilizing induced pluripotent stem cells (iPSCs) to derive an in vitro NMJ. Procedures involving the differentiation of healthy-state iPSCs into motor neurons have been optimized. The iPSC cultures were matured until day 45 after induction towards a neural ectoderm lineage, and were analyzed using immunocytochemistry, imaging, patch-clamp electrophysiology, and flow cytometry. Differentiated neurons stained positive for neuronal marker p75, Islet-1 and choline acetyl-transferase (ChAT). Electrophysiology data showed neurons were capable of high degrees of repetitive firing sequences, while flow cytometry showed neuronal purity in culture was roughly around 97%. Human skeletal muscle myoblasts have also been differentiated into myotubes-the precursors for muscle tissue-and imaged for acetylcholine receptor clusters. The culture medium was supplemented with various additives in order to determine which medium promoted greatest acetylcholine receptor (AChR) cluster density and size. It was found that AChR clusters are more prominent and enlarged when the muscle cells are cultured with Agrin supplement. Ongoing studies are looking at combining these cell types into a novel contractility assay in order to investigate NMJ function in vitro. The establishment of this system will enable more effective applications for novel therapeutics and disease progression studies.

Vascular remodeling—the modification of preexisting vasculature—often occurs in common diseases such as cardiovascular disease and cancer. Little is known about the factors controlling perivascular remodeling regarding interactions with the endothelium, and current methods of studying this phenomenon in the microvasculature have shown to be inadequate. A small-diameter, single-channel arteriole model was developed to study vascular remodeling under various flow and cellular microenvironments in vitro. The housing device consists of a plexiglass casing that allows for robust and time-efficient fabrication, attachment of flow and pressure, and in situ imaging of the vascular constructs. Type I collagen gel is embedded with human coronary artery smooth muscle cells (HCASMCs), and the lumen is formed using a needle-based subtractive molding method with a 180um acupuncture needle. The original diameter of the lumen is retained while human umbilical vein endothelial cells (HUVECs) are seeded into the lumen by hand-perfusion. Migration and realignment of both the HCASMCs and the HUVECs is observed after applying physiological flow rates of media for 3 days in co-culture. Additionally, endothelial barrier function and smooth muscle contractile function are maintained. The functionality of the model is confirmed through dextran permeability assays, calcium wave propagation, and epinephrine treatments, where dilation of the arteriole occurs upon perfusion of the hormone. These experiments demonstrate the ability of this system to increase both time and efficiency of drug studies, as well as improve understanding of microvascular diseases.

The human liver is a unique organ with the ability to regenerate quickly in response to acute injury. During this regeneration process, hepatocytes, i.e. the main cell type of the liver, receive growth factors and other cues and begin to remodel the extracellular matrix (ECM) of the tissue. Liver regeneration has been well characterized in mouse and rat models; however, human liver regeneration remains largely unstudied. In particular, the ECM remodeling process in human liver regeneration is unknown. Here, we use a humanized mouse liver injury model to study changes in the ECM over time during human liver regeneration. To study this, we implanted engineered human liver tissue "seeds" into the fat pad of FNRG-mice. These mice experience liver damage, and liver regeneration cues flood the bloodstream in response. The seeds become exposed to these cues and expand over time, mimicking human liver regeneration inside a mouse host. To study the ECM over time, we sacrificed the animals every other week and then performed special histology stains to characterize ECM components such as collagen I, IV, and fibronectin. We also immunostained for CK18/CK19, markers for hepatocytes and cholangiocytes respectively. Information on the ECM remodeling process is key to understanding human liver regeneration as a whole. Understanding this process could lead to better informed decisions regarding matrix composition in artificial human liver constructs for regenerative medicine.

Stem-cell derived engineered cardiac tissues are a promising avenue for the research of heart disease, enabling disease-specific modelling and drug screening. However, their validity as a model hinges on the similarity to native cardiac tissue, both in super- and sub-cellular structure and organization. When cultured on nanopattern substrates, myocytes orient the sarcomeres along a similar axis, allowing for greater force production and higher similarity to native cardiac tissue, though the evidence of the finer details are obscured by the diffraction limit of light. Super-resolution microscopy has opened the door to the analysis of diffraction-limited biological structures, and one such recently developed super-resolution technique, Expansion Microscopy (ExM), has made this analysis more accessible. By embedding the fluorophores from an immunostained sample into an expandable hydrogel, sub-diffraction details are physically enlarged and made measurable on standard fluorescence microscopy equipment. In this work, expansion microscopy was performed on engineered cardiac tissue to evaluate the effect of the nanopattern substrate on sub-sarcomeric structure and organization. As expected, it was found that the sarcomeres were more aligned within the cardiomyocytes, and the length between the z-lines were increased when cells were cultured on the nanopattern substrate as compared to the flat substrate. In addition, the width of the Z-disks were significantly different in nanopattern cultured cardiomyocytes as compared to myocytes cultured on flat substrate. The transverse tubules, responsible for a unified action potential, were larger in diameter and better localized with the Z-line, and the myosin heads were closely localized with the actin filament. When examined with super-resolution microscopy, the engineered cardiac tissues display sub-sarcomeric organization that allows for greater and more efficient force production, further demonstrating the efficacy of nanopattern substrates in cardiac cell differentiation and maturation. Though ExM needs further validation, it has proved a powerful sample-side tool for probing diffraction limited features.

The ability to recapitulate the dynamic presentation of signals in a stem cell's microenvironment remains a major hurdle in tissue engineering. By controlling cell growth and differentiation in 4 dimensions (i.e., time and 3D space) through biochemical and biomechanical cues, heterogeneous synthetic tissues could be produced that match the complexity of their native counterparts. By combining strategies in light-programmable hydrogels and recombinant protein engineering, we have demonstrated the ability to control the cellular microenvironment with a UV light mediated reaction that allows a covalent bond to form between a modified protein and the hydrogel. While covalent interactions form strong bonds, they are generally irreversible using mild, cytocompatible chemistries and pose potential temporal limitations. In addition, this technique uses UV light, which may be harmful to cells. Instead, by incorporating LOVTRAP into the photopatterning system, we introduce a greater amount of control by allowing for full reversibility using blue light. In the dark, LOV2 and Zdk1 dimerize to form a protein-protein bond with similar stability to an antibody-antigen interaction. Upon exposure to blue light, the dimer dissociates, allowing for fully reversible control over conjugation. A protein of interest can be tagged with the Zdk1 domain using recombinant protein engineering, while still maintaining its binding affinity for LOV2. LOV2 can then be conjugated throughout a hydrogel to make a light responsive protein binding material. By utilizing a light controlled dimerization event between proteins, the LOVTRAP system is fully reversible using only blue light. Thus far, I have been able to illustrate the photopatterning ability of LOVTRAP and am working towards demonstrating full reversibility. If successful, this technique could be used for tissue engineering applications that require mild treatment conditions and full dynamic control over the presentation of biochemical cues.

Ethics and diversity are critical components of engineering training and practice, but most undergraduate engineering programs do not address these issues in-depth. We describe the design and implementation of a novel curriculum that allows early engineering students to explore the interplay of diversity and ethics in an engineering context. Although upper-division bioethics courses may also address this relationship, we chose to implement this curriculum in a large, introductory course. We intend to provide an accessible opportunity for early engineering students, particularly underrepresented students interested in engineering, to engage with this material early and to impress upon a broad audience that diversity and ethics are critical topics in engineering. Engaging these students in meaningful discussion about the intersecting roles of diversity and ethics in bioengineering enables them to apply course competencies to their future engineering practice. Our learning objectives include: (1) Summarize key case studies regarding diversity-related ethics in STEM, (2) Identify how cultural concepts of race, gender, sexuality, and disability have shaped scientific thought (and vice versa) through history, (3) Critically evaluate literature regarding ethics and diversity in bioengineering, (4) Analyze how engineers handle implicit bias during research and design processes, and (5) Propose approaches to promote ethics and diversity in engineering practice. Course activities cover the importance of diversity and ethics competency in engineering; historic and current case studies of diversity-related ethical issues and how historical perceptions and contexts still influence modern scientific thinking and engineering design; advocacy and representation of minorities in engineering; evidence supporting the value of inclusive teaching and diverse teams; and best practices for advocacy and representation of diverse peoples in engineering. We assess the effectiveness of these teaching innovations through student surveys, student performance on assignments, and instructor observations. Additional supporting data is provided by excerpts of student work.

Influenza is a prevalent infectious disease that claims the lives of 800,000 people globally. Current diagnostic methods include cell culturing, antibody staining, and nucleic acid amplification tests that take from 4 hours to 3 days. The need for rapid, cost-effective and accurate influenza detection tests can be met by lateral flow immunoassays (LFA), which are paper-based test strips similar to pregnancy tests. Most LFAs use colored particles such as gold nanoparticles to indicate the presence or absence of a biomarker by producing a line when the biomarker is present. However, influenza LFAs require over 10 fmol of influenza nucleoproteins to produce a readable signal, which is impractical for influenza because it is present at lower levels at earlier stages of infection. There is a need for a more sensitive influenza LFA that has an improved limit of detection. Here, we investigated how replacing gold nanoparticles with quantum dots, a fluorescent label, improves assay performance when imaged with a mobile phone. We optimized our LFA by varying the concentration and volume of the reagents needed to produce a visible signal. We excited 605 nm quantum dots with a UV LED and used an iPhone SE or Nexus 5X to image the LFAs. Results showed that quantum dot-labeled LFAs imaged with cell phones had a 10x lower-limit of detection than gold nanoparticle-labeled LFAs. Limits of detection with the mobile phones of 1.5-2.6 fmol were comparable to that on a lab-based fluorescence reader, a gel imager (limit of detection of 1.9 fmol). These results suggest that cell phone imaging and fluorescent labels can be combined to make cost-effective LFAs that can be used for rapid and efficient detection in point-of-care settings.

The Oligonucleotide Ligation Assay (OLA) specifically detects point mutations in the HIV polymerase gene conferring drug resistance. Screening patients for drug-resistant HIV is a necessary step in determining the proper treatment. However, the traditional OLA is complex and lengthy, making it non-ideal for low-resource settings. We are exploiting a novel approach to adapt the OLA into a point-of-care, paper-based format. To enable downstream isothermal ligation and paper-based detection steps, the amplification step of the OLA must be designed to preferably generate many copies of single-stranded HIV DNA (ssDNA). Typically, this can be achieved using a Linear-After-The-Exponential Polymerase Chain Reaction (LATE-PCR), which uses an uneven ratio of forward and reverse primers. Though the LATE-PCR is useful for producing large amounts of ssDNA, it involves a plethora of parameters whose contributions to the reaction output are not well understood. Thus, the LATE-PCR is often optimized via trial and error and may not yield the most desirable outcome. In order to minimize wasted resources from unnecessary trials and achieve a high-efficiency amplification, we aim to understand the behavior, predict the outcome, and develop a platform for LATE-PCR optimization. We are using PCR theory and mass-action kinetics to predict exponential amplification of symmetric OLA PCR reactions. This forms the basis for a LATE-PCR model. We will incorporate the effects of key reaction parameters in this model by observing the behavior of LATE-PCR reactions when altering the number of cycles run, primer concentration ratios, enzyme concentrations, enzyme rates, salt concentrations, and primer sequences. Analysis of the model results will elucidate the effects of thermodynamics and kinetics on the LATE-PCR. Future research will focus on expanding the model of the LATE-PCR to other instrumented-free isothermal amplification approaches for a simplified point-of-care OLA.

Cells, making up every living organism on earth, are extremely complicated biological machines. Much research has been done on how cells function, including how proteins made by cells work together to create biochemical pathways necessary for growth. Recently, computational simulations of biological processes have become possible, such as in the field of systems biology, which takes a holistic view of an organism to elucidate function. Previous research has constructed an accurate "whole-cell" model of E. Coli metabolic function. But, understanding metabolic behavior is still a challenge, especially from external perturbations in the environment or an internal alteration in the form of an insertion of an extra protein. This research uses an E. Coli whole-cell model—containing carbon-dependent metabolic pathways in E. coli— to study how perturbations will affect the pathway. External changes in the resources available to the cell or an internal change to the cell’s components are simulated. In response, specific changes to the metabolic behavior of proteins will be made. These changes are made using optimization algorithms, which minimize the output of a growth “cost” function, which maximizes the growth rate of the cell. This represents the effect of evolution over time for the cell to reach an optimal level of growth. The “Tellurium” software package, using the Python programming language and created by the Sauro lab, was used for this experiment, and a collection of scripts that can be used in Tellurium were made to generalize the reproduction of this process to any model, and to visualize the results. This research can give greater insight to how cellular metabolic processes as a whole behave, and can give scientists working with in vivo experiments better predictions of the consequences of perturbations, externally or internally, on a cell.

Computational simulations of protein dynamics provide an efficient way of predicting protein behavior and are increasingly being applied to peptide and protein design. However, design tools and software focus on static structures. Incorporation of dynamics directly or through design libraries derived from dynamics simulations allows the user to focus on optimization of the native dynamics of the protein for a specific purpose. Critical libraries for design, such as side chain rotamer libraries and amino acid propensities are typically derived from static structures, which do not reflect behavior in dynamic conditions. Therefore, including rotamers and conformational propensities derived from behavior during protein simulations in molecular modeling and design software should improve the design process and outcome, particularly for peptides. Additionally, current software does not allow the user to adjust the main chain dihedral angles of the backbone according to Ramachandran plots reflecting the unique free energy landscape of each residue. The WRANGLER software was designed in order to include dynamic data to better model and design against and for dynamic systems. A number of libraries derived from dynamics simulations of all known protein folds have been incorporated. In addition, the software is interactive with a graphical interface to easily change and visualize geometries and analyze peptide/protein properties. WRANGLER was evaluated based on ability to facilitate design of several amyloid peptide aggregation inhibitors. Resulting designs were evaluated through molecular dynamics simulations for their secondary structure retention and physical properties. Control amyloid peptide aggregation inhibitors were included that have already been designed, synthesized and tested experimentally in lab. Several peptides designed in WRANGLER appear to be better than the controls by a variety of metrics. The next step is to synthesize these new designs and test them against the amyloid-beta peptide associated with Alzheimer’s Disease to see if they outperform our current compounds.
 

The cost of making new drugs is sky-rocketing: most drugs, developed using FDA-mandated animal testing, fail the approval process due to safety or efficacy concerns because animal data does not necessarily apply to human physiology. Presently, cancer biologists do not have a reliable and inexpensive tool to test the chemosensitivity of drugs on human tissue. We are developing a 3D-printed device that uses an array of biocompatible textile threads (e.g. silk or nylon) for the multiplexed delivery of drugs by capillary action in gentle physical contact with live human breast tumor samples. Fabric has three inspiring properties – biocompatibility, flexibility and low cost – which make it highly attractive for building a gentle drug delivery interface that is compatible with live tumor tissue, that can address the small sample sizes, and that can be inexpensively disseminated to clinical laboratories even in low-resource settings. We are currently optimizing mass transport through the threads and onto tissue using fluorescent drugs and dyes and plan on scaling up the device for the delivery of up to 10 different drugs (requiring up to 20 threads). Our device will allow us to compare the pharmacodynamic profiles of continuous, pulsed and sequential application of the drugs with very simple procedures. This work addresses the urgent need to develop better test assays based on intact human cancer tissue that can more closely mimic tumor physiology and predict clinical outcomes better than 2D cell culture systems and animal models.

This research aims to determine the difference between exosomes isolated from different cell culture assays. Exosomes are cell-derived extracellular particles that are present in all eukaryotic fluids and different cell culture methods may result in exosomes with different properties. This project seeks to quantify the differences between exosomes isolated from cells cultured in 2D tissue culture plates versus cells cultured in 3D porous polymer scaffolds, and these differences include: size of exosomes, exosomes yield per cell, outer membrane markers on exosomes (CD9, CD81), and total RNA contained within the exosomes. T-75 cell culture plates and poly(hydroxyethyl methacrylate) pHEMA porous scaffolds with 40 and 100 micrometer pore size are used as 2D and 3D cell culture assays, respectively, for culturing RAW 264.7 (murine macrophage) cells. Exosomes are harvested along with cell culture medium and isolated using a total exosome isolation kit. Mean particle size and exosomes concentration are measured using a NanoSight instrument and cell concentration is measured using XTT cell viability assay. Total RNA and protein contained in exosomes are isolated using total exosome RNA and protein isolation kit, and concentration of each is measured through NanoDrop spectrophotometer. The outcome of this project not only will provide a better understanding on different properties of exosomes and the effect of cell culture assay on exosomes, but also has the potential to benefit research in tissue engineering in the future.

Each year, approximately one million medical implant-associated infections occur, due to the susceptible nature of biomaterials to microorganism colonization. In an age where antibiotic resistant strains are becoming an epidemic, it is essential to develop biomaterials that do not pose an additional risk to a patient’s health. The implantation of tissue regenerative scaffolds into the body causes a foreign body response to occur, involving the encapsulation of the device, which results in macrophage differentiation. Previous research has only focused on the tissue regeneration aspects of scaffolds, but have failed to address concerns with scaffold associated infections and immune response modulation. There is a need to further understand the role of scaffolds in modulating macrophage phenotype and the effect of macrophage differentiation towards specific phenotypes on their ability to resist microbial invasions (i.e. their phagocytic abilities). We are quantifying the differentiation of macrophages into M1 (pro-inflammatory) versus M2 (anti-inflammatory) phenotypes then comparing their phagocytic abilities using an in vitro phagocytosis assay and flow cytometry. Depending on scaffold properties (pore size, construction material), implants may promote the differentiation of macrophage towards a phenotype with diminished phagocytic abilities. By quantifying macrophage differentiation and innate immune response, we can design scaffolds that will better aid the body in healing and reduce a patient’s risk of a medical device-associated infection.

Calcific aortic valve disease (CAVD) is a disease that results in aortic valve thickening and calcification, leading to valve stenosis. It is commonly experienced by aging people, especially those suffering from type II diabetes mellitus (T2DM). There is currently no effective drug therapy to prevent or treat CAVD, mainly due to the poor understanding of its etiology. The proposed study will explore the cellular and molecular mechanisms of diabetic CAVD, focusing on transcription factors Sox9 and Runx2. As the disease progresses, Sox9-expressing cells differentiate into procalcific Runx2-expressing cells, resulting in valve calcification and stenosis. We hypothesize that Sox9-expressing cells in T2DM aortic valves are reparative. To test this hypothesis, I will feed a diabetogenic high-fat diet to a mouse model susceptible to developing CAVD, which will enable genetic fate mapping of Sox9-expressing cells upon administration of tamoxifen intraperitoneally. Aortic valve sections will then be stained with X-gal, a substrate used for detection of the transgene used for the genetic fate mapping of Sox9 cells. X-gal staining will identify cells that once expressed Sox9 and immunofluorescent staining will identify Runx2 expression in cells. Quantification of Runx2+ cells with or without X-gal+ Sox9 will determine if Runx2+ cells are derived from Sox9-expressing cells and, if so, to what extent. If Sox9+ cells indeed give rise to Runx2+ cells, colocalization in staining by X-gal and Runx2 antibody is expected. This research will help identify cellular and molecular targets that may be used as a basis for future diabetic CAVD drug therapies.

2-deoxy-ATP (dATP) is a nucleotide used in DNA synthesis and its presence has been seen to improve the magnitude and rate of contractions in heart muscle cells. However, levels of dATP are naturally low in mature cells. As an attempt to develop a novel treatment for heart failure, methods to increase the expression of ribonucleotide reductase (RNR), a key enzyme in the production of dATP are being investigated. RNR is regulated by ubiquitin-proteasome degradation of the Rrm2 subunit. We constructed a variant version in which two regions were changed to prevent ubiquitination. This new variant should lead to higher levels of RNR in cardiomyocytes, which also indirectly increases levels of dATP. Our preliminary results show a successful increase in levels of both RNR protein and dATP in cultured neonatal rat cardiomyocytes. Although levels of RNR and dATP were increased, the level present in our cultured samples are much higher than expected for adult rat and mice cells. Therefore, we are currently testing this RNR variant in vitro in cultured adult rat cells, as well as in vivo in aged adult mice. These models are more representative of a therapeutic use. Preliminary results have been promising toward identifying a more effective method of increasing dATP levels for improving cardiac function.

Development of traditional anti-cancer drug has suffered from low success rate to difficulties in engineering approaches and clinical trials. Since metastasis is the major cause of cancer death, the current trend has been shifting towards a combined therapy of traditional cancer drugs and anti-migratory therapy. Cell migration assays provide straightforward visual and efficient quantitative analysis on cell migration distance and pattern. To implement these assays, one of common approach is to apply an elastomer block and create a cell-free area into which adjacent cells can migrate without damaging the cells. Our lab’s current cell exclusion migration assay is limited by uncontrolled barrier block adhesion to the substrate surface and to the well wall due to hand cutting and placement of the block. Therefore, to improve the usability and reproducibility, an accurate migration assay is developed to integrate an approach of controlling the geometry of the barrier block and confining the cell seeding area against cell penetration under the block. Our proposed barrier block fabrication utilizes laser-cut acrylic molds, which produce barrier blocks consistently in the same geometry. The block-placement device is optimized by designing a row of handles, which allows for placement of multiple blocks into the multi-well tissue culture plate at once and adjusts their positioning and adhesion precisely. Block-controlled migration is validated using a multi-well cell migration assay with respect to its ability to consistently confine cell seeding area and efficiency of use. The success of this method enables a more efficient method of anti-migratory drug screening and in vitro migration pattern analysis.