Expanded Abstracts

Measurement and Analysis of Sarcomere Length and Lattice Spacing in Cardiomyocytes after Applied Strain

Abstract: The continual improvement of healthcare technology evidently leads to a high demand for better treatment of diseases that afflict the elderly population, such as heart disease. The effects of heart disease and heart failure can be best explained and examined at the intracellular level through the analysis and understanding of changes in heart cells. The aim of this research project is to correlate acute changes in sarcomere structure to underlying substrate deformation in cardiac myocytes that are cultured on a deformable substrate. We expect that by stretching a substrate to which cardiac myocytes are attached, the cells will undergo the same amount of stretch, and consequently the sarcomere length and transverse lattice will show corresponding magnitudes of deformation. There may also be regional differences in sarcomere dimensions due to the non-uniform nature of the applied stress to the cell. The results from this type of research can be used in a high-throughput system in order to screen the effects of drugs or genetic mutations on the role of stretch in cardiac myocyte function, and may give insight into possible treatments for heart disease.

Thrombectomy Assessment Using Live-Cell Artificial Vessel

 Abstract: Thrombectomy devices are designed to retrieve blood clots in thrombosis-induced strokes. Newly designed thrombectomy devices are conventionally tested in glass or plastic tubing followed by in vivo testing. Although glass and plastic tubings provide direct lumen visualization, consistency in dimensions and ready availability, they are acellular and cannot represent the blood vessel biology. The in vivo vessels vary in dimensions and are costly, their opacity prevents observation of the device in real-time. To assess the endothelial damage induced by stroke thrombectomy, an artificial vessel was designed using low cost, optically clear and biocompatible tubular silicone substrates. The lumen surface of the substrates was treated and seeded with live bovine aortic endothelial cells (ECs). The ECs were stained using a live-cell fluorescent dye confirming the viability of the ECs before and after introducing the porcine blood clots into the lumen. This transparent artificial vessel allowed us to directly image the thrombectomy device interacting with the endothelial cells in real time. A rotational-imaging system was developed to image the whole-vessel intact followed by 3D vessel visualization. The 3D images revealed specific locations of EC damage on a thrombectomy device, providing insights and guidance to improve the devices. The degree of EC damages were quantified and normalized, showing clear differences among the six different thrombectomy devices. The degree of differences in damages among devices decreased as the diameter of the vessels increased, suggesting the importance of appropriate matching of the device to the vessel. Since iatrogenic endothelial injury may impact the salutary benefit of mechanical revascularization for ischemic stroke, this low-cost live-cell artificial vessel can be an effective method in assessing and advancing the thrombectomy device designs and procedures that minimize endothelial trauma during intra-arterial treatment.

Receptor Cleavage and Reduced CD36 Ectodomain Density on Heart and Spleen Macrophages in the Spontaneously Hypertensive Rat: A Molecular Mechanism for Defects of Cellular Binding by the Scavenger Receptor in Hypertension and Diabetes

Abstract: Patients with metabolic disease have a range of cellular defects of uncertain origin. To investigate this pathology we use the Spontaneously Hypertensive Rat (SHR) which has an increased arterial blood pressure and several cellular deficiencies. It has elevated plasma matrix metalloproteinase (MMP) activity, which cleaves the extracellular domain of multiple membrane receptors. We hypothesize that increased MMP activity also leads to abnormal cleavage of the scavenger receptor CD36. To test this, chronic pharmaceutical MMP inhibition (with CGS27023A, Novartis) of the SHR and its normotensive control, the Wistar Kyoto Rat (WKY), was used to investigate uncontrolled MMP activity on CD36. Surface density of CD36 on macrophages from the heart, spleen, and liver were determined in WKY, SHR, CGS-treated WKY (CGS WKY), and CGS-treated SHR (CGS SHR) by immunohistochemistry against CD36. The extracellular CD36 density on macrophages was found to be lower in SHR heart and spleen while higher in the liver compared to the WKY. However, SHR liver macrophages have increased CD36 density compared to the WKY. MMP inhibition restored CD36 density on SHR cardiac and splanchnic macrophages to WKY levels. Culture assays with murine macrophages (RAW 264.7) after incubation in WKY or SHR plasma were used to test for adhesion of light-weight red blood cells (RBC) to CD36. RBC macrophage adhesion was reduced after SHR plasma incubation compared to WKY plasma. Western blot media analysis also shows higher CD36 extracellular protein fragment levels following exposure to SHR plasma. MMP inhibition in the SHR plasma increased RBC macrophage adhesion and decreased receptor fragments in media compared to untreated plasma. In conclusion: MMP activity in SHR plasma causes cleavage of CD36 receptors, which attenuates cellular functions. MMP inhibition restores CD36 density and cellular dysfunctions in the SHR. MMP blockade and receptor cleavage remains to be tested clinically.

Static stretch affects neural stem cell differentiation along the oligodendrocyte lineage

Abstract: Neural stem and progenitor cell (NSPC) fate is strongly influenced by mechanotransduction. In particular, modulation of substrate stiffness affects differentiation of neurons and astrocytes from rat NSPCs. Thus, mechanical cues such as force are tightly linked to NSPC fate and differentiation, suggesting that these cells efficiently alter their behavior in response to physical forces in a mechanosensory manner. In order to test whether NSPCs are sensitive to another mechanical stimulus, stretch, we built a device designed to deliver a 10% static equibiaxial strain to cells cultured on flexible silicone elastomer membranes and studied the effect of stretch on mouse NSPC differentiation. NSPCs derived from cerebral cortices of embryonic day 12.5 mice were cultured and differentiated on unstretched, stretched, and pre-stretched membranes. The pre-stretched membrane provided a substrate of equivalent stiffness to the stretched membrane but did not deliver a stretch stimulus to the cells in order to distinguish the effects of stiffness and stretch. We found that static stretch greatly impacts NSPC differentiation patterns in a manner independent from substrate stiffness and that differentiation into oligodendrocytes, but not neurons or astrocytes, was significantly affected by stretch. Further, stretch effects are mediated by integrin-ECM linkages. Our data demonstrate the direct role mechanical forces such as stretch play in dictating the lineage choice of NSPCs. By obtaining a deeper understanding of how NSPCs respond to their mechanical environment we can design biomaterials with optimal properties for NSPC transplants to treat neurological disorders.

Evaluation of collagen hydrogel preparation method on embryonic stem cell behavior

Abstract: We have been developing encapsulated and topographic 3D collagen-based hydrogel models to image in situ the performance of materials and behavior of embryonic stem cells during early induction with retinoic acid. For the encapsulated model, the cells were polymerized within hydrogels assembled from either 2 g/l or 4 g/l initial collagen concentrations. For a topographic model, cells were placed on top of the 2 g/l hydrogels cross-linked with carbodiimide (EDC), EDC/N-Hydroxysuccinimide (NHS) or genipin and separately on the 2 g/l and 4 g/l 37 °C or on the 2 g/l and 4 g/l 27 °C assembled materials. The cells encapsulated or placed on top of 4 g/l hydrogels extended slower compared to cells interfaced with 2 g/l materials. Initial collagen concentration and the cross-linking both affected the embryonic stem cells’ behavior in a topographic model. On top of unmodified 2 g/l 37 °C collagen hydrogels, the cellular extension was concomitant with migration and subsequent aggregation into clusters. The collagen of the supporting extracellular matrix aligned at the periphery of the formed cluster. On top of the cross-linked 2 g/l materials, the extended stem cells remained scattered. For the materials modified with EDC, the cells aligned collagen fibers while making a clearance while no fiber alignment was observed for the EDC/NHS and genipin modified gels.

Preclinical In-Vivo Evaluation of Pulsed Dye Laser and Photodynamic Therapies on Normal Vasculature

Abstract: Pulsed-dye laser (PDL) therapy is the gold standard for treatment of port wine stain (PWS), but complete removal is infrequently achieved. Photodynamic therapy (PDT) is under study as an alternate protocol. Our previous data suggest that a protocol with PDT and PDL irradiation at reduced light doses can achieve persistent vascular shutdown. Our study objective is to evaluate this PDT/PDL protocol with use of the talaporfrin sodium (NPe6) photosensitizer. For PDT, we activated intravascular NPe6 with laser irradiation (wavelength = 664 nm). PDT was followed immediately by PDL irradiation (10 mm, 3-7 J/cm2, 585 nm). We used the dorsal window-chamber model and laser speckle imaging to monitor blood-flow dynamics. In this study, we defined a successful treatment outcome as achieving persistent vascular shutdown within the window, seven days following PDT/PDL treatment. Dose-response analysis enabled us to identify characteristic radiant exposures for PDT and PDL alone that achieved vascular shutdown. We identified specific classes of responses in the combined PDT/PDL treatments: no vascular shutdown, acute vascular shutdown followed by gradual restoration of blood flow, and acute vascular shutdown that persisted over the seven-day monitoring period. Our preliminary data suggest that the PDT/PDL therapy is a viable treatment option for PWS vasculature.

Macrophage form and function on micro- and sub-micropatterned titanium surfaces

Abstract: In response to cues in the microenvironment, macrophages polarize into either inflammatory phenotype to fight infection or anti-inflammatory phenotype to facilitate tissue repair process. Although many studies have elucidated about how soluble cues influence their phenotype, relatively few have focused on how insoluble cues in the microenvironment regulate their behavior. We recently demonstrated that macrophage cell shape influences their phenotype. Specifically, macrophage forced to elongate on micropatterned lines polarized towards an alternatively activated, pro-healing phenotype. In this study, we explore how surface topology might be leveraged to alter macrophage cell shape and phenotype. We fabricated micro- and sub-micropatterned titanium surfaces using deep etch techniques to contain grooves, which have been previously shown to promote cell elongation. Bone marrow derived macrophages were seeded on titanium surfaces with grooves of varied geometries, ranging from 150 nm to 50 µm. We found that macrophages elongated along the grooves of the titanium substrates in a biphasic manner, where the highest degree of elongation was observed on surfaces with grooves of approximately 450-500 nm wide. Expression of Arginase-1 (Arg-1), a marker of the pro-healing phenotype, was also dependent on groove size. At very small groove widths, expression of Arg-1 was low, but larger groove sizes an increase in Arg-1 expression. Thus, we confirmed that surface grooves on titanium surfaces influence macrophage elongation, and find that surface topographies might also drive macrophages to polarize towards an anti-inflammatory phenotype. Current work is focused on examining macrophage cytokine secretion by ELISA, and expression of other phenotypic markers by FACS. We believe that a better understanding of how surface topography modulates macrophage phenotype will allow the engineering of materials effectively control the foreign body response to biomaterial implants.

Polymer-Templated Titanium Dioxide Nanostructures for Study of Cell and Bacterial Response

Abstract: Titanium is the material of choice for medical implants. Understanding of cell response to nanostructured titanium is limited by control of both the chemical and physical properties at the oxide surface. Additionally, no current technique can generate well-defined 10-100 nm titania features, of particular interest for cell and bacteria studies. We have developed a unique strategy for precise, independent control of TiO2 nanostructure and chemistry. Using nanoimprint lithography, we build nanostructured polymer substrates upon which we deposit a conformal, 5 nm-thick amorphous titanium oxide layer with ALD. As confirmed by SEM and AFM, the nanostructures are topographically unchanged by TiO2 deposition. Our technique ensures that both surface chemistry and nanotopography can be controlled across all experimental conditions. Thus we can study physical mechanisms dictating cell response to very small nanostructures, often at size scales rivaling cells’ adhesion proteins. Lines, (height=100 nm; periodicities=140-800 nm) as well as arrays of posts (diameter= 160 nm; periodicities= 600, 700 nm) were compared with flat controls and corresponding PMMA substrates. Primary murine macrophages seeded on TiO2 coated, nanolined substrates 1) align with the grating and 2) elongate compared with flat controls. On TiO2 coated posts, they spread in a more equiaxed manner compared with controls. Compared with PMMA nanolines, macrophages seeded on TiO2 nanolines elongated more drastically. This result highlights the importance of surface chemistry in mitigating cell response. Thus, preliminary studies of the TiO2 coated nanostructures show promise in inducing a pro-healing macrophage response and may also reduce bacterial adhesion. The methodology described here yields a versatile platform for the mechanistic study of cell response to arbitrary, precisely controlled nanostructures that can be modified to display any desired surface chemistry.

Electrotactile and audio feedback device for evaluation and correction of gait asymmetry in stroke patients

Abstract: A common impairment after stroke is increased asymmetrical weight bearing between the two limbs while standing and walking. Restoring symmetry of gait and posture is an important goal of stroke rehabilitation. The objective of this project is to develop a device: 1) to evaluate posture (static task) and gait (dynamic task) asymmetry in stroke population, and 2) to provide real-time feedback to correct it. The device will consist of force sensors (placed on the insoles – on the heel and the ball of each foot), an electro-tactile feedback unit (on the front thigh and back), an Arduino microcontroller, and a control interface with audio feedback. Static task asymmetry will be measured by weight bearing force on each foot, while dynamic task asymmetry will be measured by the length of stance time on each foot, in addition to the static measures. Real-time feedback will be provided when posture or gait asymmetry is detected based on the sensor information. If weight or time spent on one leg exceeds a set threshold, a noxious electric stimulation will be generated on that limb in order to stimulate a weight shift to the opposite limb (static task) or to reduce the stance time in the same limb (dynamic task). An audio feedback in the form of a graduated beep will also be used as an additional feedback. The design and testing of the device will be reported.

Biocompatibility and thermal evaluation of transparent yttria-stabilized-zirconia (YSZ) cranial implants

Abstract: The goal of the Windows to the Brain (WttB) is to improve patient care by providing a technique for delivery and/or collection of light into/from the brain, on demand, over large areas, and on a chronically-recurring basis without the need for repeated craniotomies. WttB holds the transformative potential for enhancing the light based diagnosis and treatment of a wide variety of brain pathologies including traumatic brain injury, stroke, and brain tumors. To evaluate the functionality of yttria-stabilized-zirconia (YSZ) cranial implant for optical therapy and imaging, in vivo biocompatibility and the thermal profile of the tissue underneath the implant were studied. Biocompatibility was studied by implanting YSZ on hamster dorsal skinfold chamber and monitoring host tissue response by transillumination and fluorescent microscopy for two weeks. The thermal functionality of YSZ for 810 nm laser was investigated in vitro by comparing the surface temperature of YSZ implant and mouse skull flap when they were both covering a 37 C agar gel brain phantom. For the biocompatibility study, we compared leukocyte activation, blood vessel diameter, blood flow rate, and vascular permeability as a response to YSZ, commercially available cranial implant materials and control sample (tissue underneath the glass slide). The results for tissue response to YSZ indicated minimal inflammatory reaction of the host tissue at the microscopic level over 2 weeks which was comparable with commercially available cranial implants. The results of thermal profile showed that the thermal elevation in the brain phantom underneath the implant was less than 0.5 C, meaning that routine optical imaging and therapy procedures can be performed without inducing thermal damage to the brain covered with YSZ. The results suggest that given the biocompatibility and thermal response, YSZ implants can potentially allow for safe and feasible chronic non-invasive optical imaging and therapy of brain.

Development of a Material Platform for Hypoxia-Targeted Gene Delivery

Abstract: Gene therapy represents a promising approach for the treatment of ischemic diseases, however efficient delivery remains a challenge. We aim to improve delivery efficiency by developing polymeric systems capable of targeting the hypoxic character of ischemic tissue. Our approach involves the addition of a hypoxia-sensitive compound, 2-nitroimidazole, onto the polymeric backbone of chitosan. We hypothesized that chitosan carriers functionalized with 2-nitromidazole would demonstrate increased accumulation and transfection efficiency in hypoxic cells. Chitosan was modified with a synthesized derivative of 2-nitroimidazole and characterized with 1H NMR. Uptake studies using FITC tagged chitosan for both HMVEC and HEK293 cells demonstrated dose-dependent endocytosis, with decreased uptake in hypoxia. A decrease in the percentage of FITC positive cells over several days indicates that the novel polymer is slowly exocytosed. The modified polymer demonstrated increased transfection efficiency in normoxia and hypoxia. During an in vivo pilot study, Alexa Fluor 750 tagged chitosan was administered to ischemic and normal murine hindlimbs, and the ratios of hindlimb radiant efficiencies were taken over two days. A slower rate of clearance in the ischemic limb was observed for modified chitosan compared to control unmodified chitosan. This initial data indicates that the modification of chitosan with 2-nitroimidazole does not adversely affect uptake and increases transfection efficiency. In vitro and in vivo results have indicated that chitosan remains intracellular for extended periods of time both in normoxia and hypoxia. However, a slower clearance of modified chitosan from the ischemic hindlimb suggests that 2-nitroimidazole may affect intracellular trafficking in hypoxia. In order to improve upon selectivity for hypoxia, future work will include higher degrees of substitution and selecting for chitosan formulations with increased baseline exocytosis.

Multivalent antibodies built on DNA scaffolds to modulate cell signaling and function

Abstract: Cellular receptors frequently assemble into higher order oligomeric complexes that function cooperatively and can synergize with other receptor types for enhanced affinity, selectivity and noise filtration, thereby modulating the biological outputs in response to external inputs. We have recently exploited a new class of one-dimensional DNA molecules synthesized by an efficient isothermal enzymatic reaction called rolling circle amplification (RCA), utilizing nucleic acids as modular building blocks for the development of multivalent synthetic ligands to target specific cell receptors. In this study, DNA molecules synthesized by RCA and containing biotinylated deoxyuracil could serve as a polymeric scaffold on which to immobilize antibodies in a defined and programmable manner to interrogate and modulate cell receptor functions. Our model cell signaling system was apoptosis resulting from CD20 clustering in B cell cancers, beginning with intracellular influx of calcium ions (Ca2+), subsequent phosphorylation of phospholipase C gamma 2 (PLC?2), and activation of caspases to culminate in induction of apoptosis followed by necrosis. We found that the multivalent anti-CD20 Poly-Ab was significantly more effective in binding to and clustering CD20 receptors and inducing apoptosis of target Ramos cancer B cells than the monovalent counterpart (Mono-Ab). Our multivalent DNA material approach represents a new chemical biology modular tool to interrogate cell receptor signaling and functions. For instance, the length of the RCA backbone and ligand density can be easily modified by altering RCA reaction parameters such as incubation time and derivatized nucleotide incorporation. In addition, our system has great potential to incorporate multiple types of ligands (e.g., rituximab and ofatumumab monovalent anti-CD20 antibodies, aptamers or carbohydrates) that may target different

Transmucosal Delivery of Celecoxib into Oral Cancer Lesions from Multifunctional Nanodiamond Constructs

Abstract: Celecoxib (Cxb) is an insoluble drug with the potential to treat oral cancers. However, Cxb administration via ingestion results in low on-site bioavailability and high systemic toxicity. Direct injections are traumatic and inconvenient. To overcome these challenges, sustained and localized delivery of Cxb into oral cancer lesions is required. However, Cxb molecules are unstable in the aqueous oral cavity. Another challenge to overcome is the protective mucus layer that covers the oral mucosa and traps most particulates. To facilitate the release of Cxb into oral cancer lesions, we propose an adhesive, biodegradable patch embedded with mucopenetrating nanodiamond-Cxb complexes. Nanodiamonds (NDs) are carbon nanoparticles that act as effective drug carriers due to their functionality, biocompatibility, and drug sequestration capability. Our NDs are polymer-functionalized to allow for mucosal penetration. We are currently investigating the mucopenetrative efficiency of our multifunctional ND construct with Franz diffusion cell experiments and in vitro cell models. Furthermore, we are generating Cxb loading and release profiles for our ND construct with UV spectroscopy. Preliminary cell viability assays conducted by treating oral cancer cells lines with ND-Cxb complexes have also shown that ND-Cxb may be better at killing tumor cells than free Cxb. Overall, our mucopenetrating ND-Cxb platform can overcome the barrier nature of mucous membranes, serving as a model for nanoparticle-mediated transmucosal drug delivery.

Nanopatterning of polymer surfaces for controlled bacterial and mammalian cell adhesion

Abstract: Our objectives are to develop polymer surfaces that could control cell adhesion solely by altering physical surface topography and to study the role of geometric parameters on adhesion. We used nanoimprint lithography to create line gratings (width = 134 ± 9 nm), and pillared surfaces (diameter = 215 ± 9 nm, spacing = 342 ± 22 nm) on polymethylmethacrylate (PMMA) films. In the bacteria study, we plated E. coli and observed that after 20 hours, bacteria on the flat film and line gratings are fully rod-shaped, the normal E. coli cell morphology. On round pillared surfaces, bacteria appear more deflated on pillars, stretching over the spaces between the pillars. In live dead assays, we observed decreased bacterial adhesion and viability on the nanopillars in comparison the flat PMMA and line gratings. In another study, we seeded primary murine macrophages onto PMMA films. Previous work showed a correlation of cell shape and macrophage phenotype in that elongated macrophages adopted the pro-healing phenotype (McWhorter et al. 2013). After 24 hours, we observed that the macrophages adopted different cell morphologies, possibly indicating changes in their phenotype. Macrophages showed a spread-like morphology on the flat film. On line gratings, macrophages elongated and aligned in the direction of the lines. Macrophages on round pillars were more equiaxed. Both studies demonstrate that imprinted nanostructures in the 100-500 nm-size range could induce various mechanisms that led to the materials’ antibacterial properties as well as the modulation of macrophage response toward pro-healing versus pro-inflammatory.

Alginate Encapsulated Islets Allow For Adequate Tissue Oxygenation Even At Hypoxic Conditions

Abstract: Post-transplant graft failure and death is a serious setback negatively impacting human islet allotransplantation success. This has been postulated to result from hypoxia encountered immediately after transplantation due to poor implant-site vascularization. In order to survive and function adequately, transplanted islets must be able to withstand the transient hypoxic shock until they are adequately vascularized. In this study, we evaluated the effects of in vitro hypoxic culture on islet viability and function in alginate bioencapsulated islets at predetermined oxygen concentrations for a three day period. 10,000 IEq of islets isolated from young Yorkshire pigs were cultured for 7 days in vitro in UCI maturation media (n=3). 5000 IEq of islets were encapsulated in 3% UP LVM alginate. The other 5000 IEq were left unencapsulated (control). The islets were then incubated in a hypoxia chamber at 0%, 2.5%, 10% and 20% O2 for 72 hours. At 24 and 72 hours after incubation, 200 IEq of islets were stained with fluorescent viability (Calcein Blue/Propidium Iodide) and apoptosis dyes (YoPro-1) and examined under a fluorescent microscope. Encapsulated and free islets maintained viability (92±1; free, 85±3; encapsulated) best at 20% O2. Encapsulated islets remain viable at anoxic conditions (0% O2), but this trend could not be seen in other low oxygen environments (4±1%; 2.5% O2, 14±2%; 10% O2). No significant additive detrimental impact on islet viability was noted in alginate encapsulated islets cultured at hypoxic conditions when compared with unencapsulated islets (12±2; free, 13±1;encapsulated), indicating no compromise in their oxygen permeability. This study demonstrates that alginate encapsulated islets are able to allow for adequate tissue oxygenation even at hypoxic conditions and are ideal candidates for future studies, where specific strategies targeted at ameliorating hypoxic injury to islet grafts during the immediate post-transplant period will be evaluated.

 

Optimization of an auxetic flow diversion stent for perforator patency

Abstract: Flow diverters have recently shifted the perspective on treatment goals for hemorrhagic stroke. These densely-braided mesh cylinders are endovascularly deployed within an artery across the neck of a cerebral aneurysm to reduce the risk of rupture. The low porosity of these devices alters the local fluid shear profile so that flow passes longitudinally through the stent rather than radially into the aneurysm sac. That the device is porous is also itself significant, as this is the basis for maintaining patency in perforating arteries, which experience a pressure gradient that continues to drive blood flow. Unfortunately, a growing body of adverse clinical reports suggests that the mechanical properties which enable this selective flow diversion are highly sensitive to deployment technique and vascular morphology. Many hold that these issues are tied to the device’s braiding-based fabrication method, currently the only commercial scale technique capable of producing features of the required size. Titanium deep-etching (TIDE) offers a promising solution to this problem, enabling orders-of-magnitude in improvement upon machining resolution and consequently allowing for the employment of micro-scale design principles to subvert some traditional limitations of bulk material properties. One such design technique has led to the development of auxetic materials, which effectively exhibit negative Poisson’s Ratios. This work aims to couple these design and fabrication approaches to explore how the behavior of this class of material can be tuned to enhance the flow diversion effect.

SOFTWARE-BASED DESIGN AND FABRICATION OF COMPLEX 3D SHAPED MICROPARTICLES

Abstract: Complex three dimensional microparticles could play an important role in several emerging bioengineering technologies. For example, advanced drug delivery relies on physically tailoring the shape of microparticles to maximize transport or uptake by specific cell types. Particle fabrication in microfluidic devices provides several advantages, including high throughput production and controllable composition of particles. However, the shape of particles is restricted to certain types, like spheres, ellipsoids, and extrusion of 2D patterns. To aid in these application areas one needs to develop an easy design approach for complex particle shapes with the ability for rapid iteration. We demonstrated a fabrication method based on engineering the flow stream inside microchannels (300×1200µm) to generate microparticles with two extrusions, parallel and perpendicular to the flow (Fig. A, B). The ultimate goal is developing complex 3D shaped microparticles using software-based design. We use inertial flow deformation to shape a precursor stream allowing for complex 3D shaped particles. The cross-sectional shape of the inertial sheath flow is sculpted using non-reversible displacement induced by micropillar structures. In the region the pattern is formed after the last micropillar, UV illumination is applied on the deformed stream through a shaped mask, shown in the circular inset of Fig. B. It is important to note that only the main stream of the sheath flow includes the photoinitiator, so the stream can be trimmed to be the shape of the mask. The cured microparticle is formed by the intersection of the deformed flow stream and the path of UV illumination. The expected particle shape is shown from three different points of view in Fig. C, while the image of the microparticle is presented in Fig. D. The comparison illustrates the ability of the fabrication system to generate complex 3D microparticles with high quality.

Vascular Stents with Rationally-Designed Sub-Micrometer Scale Surface Patterning

Abstract: Drug-eluting stents have revolutionized the field of interventional cardiology by reducing incidence of restenosis through local delivery of drugs that inhibit inflammation caused by implantation-induced injury. However, growing evidence suggests that this may also inhibit reestablishment of the endothelium, thus delaying healing and increasing potential for thrombogenic stimulus. Herein, we discuss our recent progress towards realization of next-generation titanium (Ti) stents that seek to mitigate adverse physiological responses to stenting via rational design of stent surface topography at the micro- and nanoscale. Specifically, we discuss: 1) advances which now allow patterning of features in Ti substrates down to 150 nm, which represents the smallest features achieved to date using our novel Ti deep reactive ion etching (Ti DRIE) technique; 2) creation and evaluation of balloon-deployable, cylindrical, surface-patterned stents from micromachined planar Ti substrates; and 3) integration of these processes to produce a device platform that allows, for the first time, evaluation of surface patterning in more physiologically relevant contexts, e.g. in vitro organ culture. Collectively, these efforts represent key steps towards our long-term goal of developing a new paradigm for stents in which rationally-designed surface patterning provides a physical means for complementing, or replacing, current pharmacological interventions

Optimzing the Performance of a MEMS-Based Mechanoporation Device for Ultrahigh Throughput Cellular Manipulation

Abstract: Numerous methods exist for introducing exogenous materials into cells en masse by physical membrane poration (e.g. electroporation, sonoporation, and others). However, most are limited by risk of cell damage, thus raising safety concerns for therapeutic applications. Recently, we reported a new method for ultrahigh throughput (UHT) cellular manipulation via mechanical membrane poration, i.e. UHT mechanoporation. This concept relies upon a microelectromechanical systems (MEMS) functional core composed of cell capture sites with monolithically integrated, sub-micron scale solid penetrators. Negative flow through aspiration vias at the bottom of the capture sites draws cells onto the penetrators, causing membrane poration. Cells are then released by reversing flow through the vias. The transient nature of the poration enables transfection via diffusion-driven influx of exogenous molecules, while massive parallelization provides potential for UHT operation (e.g. 10k capture sites). However, while our original studies validated concept feasibility, low poration efficiencies were observed (~8%).In this presentation, we describe results from studies focused on optimizing our UHT mechanoporation device. These studies have been made possible through implementation of high-resolution fluorescence imaging and real-time pressure drop measurement during device operation. We find that the low poration efficiencies observed in our earlier studies may have been caused by insufficient flow rates during the cell capture and penetration steps. Moreover, inadequate washing could have diluted the proportion of porated cells in the collected population. Finally, potential for incomplete cell sealing over the capture wells, due to their slight undersizing relative to the wells, may have reduced the force imposed on the captured cells. Collectively, these results have demonstrated need for further study and have helped identify directions for our ongoing optimization efforts.

Demonstrating Compatibility of a Chitosan/Alginate Enteric Coating for Targeted Oral Drug Delivery of Nanoparticles

Abstract: In the treatment of inflammatory bowel disease (IBD) and colon cancer, targeted drug delivery is desirable as it can minimize side effects by reducing drug delivery to healthy tissue. One general strategy is to conjugate protein targeting ligands to the surface of a delivery vehicle to increase the residence time at the target site. Unfortunately, the gastrointestinal tract poses major problems for proteins during oral drug delivery, such as acidic denaturation and enzymatic degradation, and therefore, the delivery system needs to be protected by what is referred to as an enteric coating. However, most enteric coatings require the use of an organic solvent during the coating process which would denature any surface conjugated proteins. To circumvent this problem, we adapted a layer-by-layer chitosan/alginate coating process for our protein conjugated nanoparticles. Alternating zeta potential measurements indicate that the process of coating the delivery vehicle is not hindered by the presence of surface proteins. Additionally, we demonstrate that this coating delays drug release in a manner that is dependent on the pH of the environment. Finally, we demonstrate through cellular binding studies that the targeting ligands maintain functionality after exposure to a basic pH environment (representative of the colon), suggesting that the enteric coating completely sheds from the delivery vehicle. Taken together, these results demonstrate that our enteric coating process could be used to develop targeted delivery systems for the oral treatment of colonic diseases.

Engineering Biomaterials for Passive and Active Immunomodulation

Abstract: To mitigate a chronic inflammatory host response, engineers often choose an inert biomaterial to limit unwanted effects on the immune system. Poly(ethylene glycol)(PEG) is one such material used to passively modulate immune activation. PEG has the unique ability to tightly associate with multiple water molecules per monomer, attributed to the hydrophilicity and charge distribution across the polymerized macromer, and is thought to render the material “invisible” to the body. Additionally, PEG is non-toxic, water soluble, and easily modified, making this polymer one of the most useful biomaterials in the field today. Unfortunately, PEG still elicits an undesired inflammatory response from macrophages, characterized by release of cytokines TNF-a and IL-1ß. Previous studies in our laboratory demonstrated that coating a biomaterial with a protein that actively decreases this cytokine release profile can increase the biocompatibility of a biomaterial. CD200, a membrane glycoprotein whose receptor is found exclusively on myeloid cells, is one such anti-inflammatory protein. This self-recognition molecule sends an inhibitory signal when bound to CD200R on macrophages, thus diminishing TNF-a and IL-1ß expression. We proposed to combine passive (PEG) and active (CD200) immunomodulatory mechanisms for further control of biomaterial compatibility. Through confocal microscopy we show evidence of CD200 immobilization onto a PEG hydrogel and ultimately demonstrate that these CD200-functionalized PEG hydrogels not only decrease the secretion of inflammatory cytokines in BMDMs, but also limit the inflammatory response in vivo up to 24 hours.

Nanoscale substrate features modify multispecies biofilm dynamics

Abstract: Treatment of medical biofilm infections requires high doses of antibiotics and often the infection is prone to relapse. Therefore, a persistent method for long-term biofilm inhibition or dispersal is essential. Pseudomonas aeruginosa is an opportunistic, biofilm-forming pathogen associated with lung infections in cystic fibrosis and in indwelling device infections. Many such infections results from multispecies biofilms, which develop and are maintained through chemical signaling and metabolite exchange. Here we demonstrate control of a co-culture biofilm composed of E. coli and P. aeruginosa, which exhibit an antagonistic interaction, through purely physical cues from periodic structures on growth substrates. P. aeruginosa produces signaling molecules which E. coli recognizes and which trigger a dispersal response in E. coli biofilms. However, E.coli biofilms grown on periodically microstructured substrates resist this dispersal response, and the height of the substrate features modify the species composition of E. coli cells retained in the co-culture biofilm. Substrate features modulated the biofilm composition from complete P. aeruginosa biofilms to complete E. coli biofilms and a continuous variation of volume fractions in co-cultures. The mechanism for switching signaling pathways in the co-culture is proposed to be through alteration of biofilm morphology and subsequent metabolite accumulation in biofilms grown on periodic structures. Furthermore, such substrate features are persistent, unlike surface chemistry modifications or antibiotic leaching strategies for biofilm resistant surfaces, and are thus promising for long-term control of biofilm structure and properties. As an example, we use these structured substrates, to produce probiotic surfaces which resist P. aeruginosa biofilm formation for more than two weeks. Moreover, these surfaces rendered the resulting biofilm more susceptible to antibiotic treatment than co-cultures grown on flat surfaces.

LLLT and Pharmacological Approaches for Wound-Healing in Cell Models

Abstract: Low light level therapy (LLLT) has been used to increase wound-healing, but challenges in dosimetry and other treatment parameters have limited this approach; moreover, both positive and negative results have been reported. Using in-vitro wound healing assays; we demonstrate effectiveness of two different LLLT sources, a clinical laser (bioLITEC) and an LED array (Celluma). The Celluma emits light ranging from 465-901 nm, is flexible allowing treatment of multiple wound areas, offers continuous or pulsed delivery, and is highly portable and user friendly. Most cold laser systems are large and require advanced expertise to operate. Both sources were effective at 637/652 nm and 806/901 nm in three different cell types: PtK2 rat kangaroo renal epithelial cells, U2OS human osteosarcoma cells and A549 adenocarcinoma human alveolar epithelial cells. The LED performed at least as well as, and, in some cases statistically better, than the clinical laser. We have shown that a new nitric oxide donating drug nitrosyl-cobinamide (NO-Cbi) accelerates wound healing in the cell model systems described above. When LLLT is combined with NO-Cbi treatment, the rate of wound-healing is greater than with either the pharmacological or LLLT treatment alone. In conclusion, we report, (1) LED-stimulated wound healing which performs as well as a clinical laser system, and (2) a combined LLLT plus pharmacological therapy for wound-healing that out-performs either modality alone.

Abstract Title: Microstructural features of the collagen hydrogels prepared with different ions are revealed by second harmonic generation (SHG) imaging

Abstract: The interactions of ions and other charged species are known to modulate proteins’ solubility and aggregation processes. Contrasts that uniquely identify aggregated structures within the biologically derived protein scaffolds could help to clarify these modulations and aid in scaffold engineering. Employing second harmonic generation (SHG) contrast we discovered that added phosphate induces an increase in the aggregated collagen structures’ (fibers) length concomitantly leading to micro-structural heterogeneity within the 3D hydrogels. Specifically, the 1 µm in length collagen fibers formed in 30 mM phosphate-only buffer, 37 °C and 2 g/l collagen solid content extend to 45 µm and increase in width in high (>/=60 mM) phosphate. The effective pore size within hydrogels also increases from about 5 µm in the hydrogels formed at 30 mM phosphate to over 24 µm in the hydrogels formed at 160 mM phosphate. Additionally, the contributions of sodium chloride will be discussed within this context. Our studies remove guesswork from engineering collagen-based hydrogels for various applications.

 Abstract Title: Multi-scale modeling of the pH-controlled self-assembly of biomaterials from peptide amphiphiles

Abstract: Self-assembling nanomaterials that can change shape or structure in response to environmental stimuli hold a great promise to revolutionize medicine and biotechnology. However, current discovery is slow and often serendipitous; therefore, there is a great need in detailed, predictive modeling. Here we report a multi-scale modeling study based on two state-of-the-art techniques to elucidate pH-dependent morphological transition between spherical micelles and cylindrical nanofibers that are self-assembled from peptide amphiphiles (PA), palmitoyl-I-A3E4-NH2. The coarse-grained molecular dynamics simulations revealed that self-assembly of spherical micelles is driven by hydrophobic interactions between alkyl tails when the electrostatic repulsion is strong. Merging of these micelles into nanofibers is driven by ß-sheet formation between the peptide segments when the electrostatic repulsion is weak. The all-atom constant pH molecular dynamics simulations revealed a cooperative transition between random coil (as exhibited in micelles) and ß-sheet (as exhibited in nanofibers) in the pH range 6–7, in agreement with experiment. Interestingly, although the bulk pKa is more than one unit below the transition pH, consistent with experiment, the highest pKa’s coincide with the transition pH, suggesting that the latter may be tuned by modulating the pKa’s of a few solvent-buried Glu sidechains. Taken together, the present study illustrates a multiscale modeling strategy that may aid in the design and discovery of pH-responsive nanomaterials.

Abstract Title: Development of targeted systemic treatments for metastatic prostate cancer using engineered ligands

Abstract: Metastatic prostate cancer has proven difficult to treat with a 5-year relative survival of only 28%. Once prostate cancer has spread to other areas of the body, systemic treatment is needed. Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to anti-cancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. This research investigates the use of two targeting ligands: an engineered version of the serum protein, transferrin, and the minibody, A11. The engineered transferrin and A11 have been designed to enhance cellular association, targeting the transferrin receptor and prostate stem cell antigen, respectively, which are overexpressed on metastatic prostate cancer cells. These ligands have shown success targeting cancer cells in vivo. A11 has been used as a PET imaging agent for prostate cancer detection, and a molecular conjugate of the engineered transferrin successfully eliminated tumor cells in a local intratumoral glioma model and outperformed the wild-type transferrin molecular conjugate. We want to translate the success of the A11 minibody and the engineered transferrin into a targeted drug delivery vehicle for systemic treatment of metastatic prostate cancer. Cellular association studies with our engineered transferrin demonstrate that it would be more effective than the wild-type transferrin in delivering drugs to prostate cancer cells. Similar studies performed with A11 also indicate that it would be successful as a targeting agent for prostate cancer cells. To allow for targeted systemic drug delivery, nanoparticles were conjugated with A11, which resulted in enhanced cellular uptake of the PEGylated nanoparticles. Currently, we are investigating the effects of targeted nanoparticles loaded with anti-cancer drugs.

Abstract Title: Biomimetic Camouflage Inspired By Cephalopods

Abstract: Cephalopods are known as the chameleons of the sea – they can alter their skin’s coloration, pattern, texture, and reflectivity to blend into the surrounding environment. Despite much research effort, there are few known strategies (natural or artificial) for emulating the unique dynamic reflectivity and coloration of cephalopods. We have drawn inspiration from self-assembled structures found in cephalopods to fabricate tunable biomimetic camouflage coatings. The reflectance of these coatings can be dynamically modulated between the visible and infrared regions of the electromagnetic spectrum in situ. Our studies represent a crucial step towards reconfigurable and disposable infrared camouflage for stealth applications [1].[1] L. Phan, W.G. Walkup IV, D. D. Ordinario, E. Karshalev, J-M. Jocson, A. M. Burke, A. A. Gorodetsky. Reconfigurable Infrared Camouflage Coatings from a Cephalopod Protein. Adv. Mater. 2013 25, 5621.
Abstract Title: Bulk Protonic Conduction in a Cephalopod Structural Protein

Abstract: Despite showing great potential, protein-based protonic conductors remain largely unexplored even though numerous examples of natural and artificial proton conductors currently exist. Here, we report both the discovery and characterization of bulk protonic conduction in reflectin, a protein originating from cephalopods. Reflectin, in the solid state, features electrical figures of merit which compare favorably to those of artificial protonic conductors. These excellent figures of merit have enabled the first example of a protein-based protonic transistor. These findings represent an important step towards the next generation of fully biocompatible, protein-based proton conducting materials.

Abstract Title: New compstatin peptides containing N-terminal extensions and non-natural amino acids exhibit potent complement inhibition and improved solubility characteristics

Abstract: The complement system provides a first line of defense against pathogens in the bloodstream. Complement activation leads to cleavage of complement component C3, which ultimately leads to phagocytosis, inflammatory response, and cell lysis. While complement has evolved toward rapid recognition and elimination of pathogens, the complement cascade must be tightly regulated to confer protection of host cells and tissues against complement-mediated damage. Breakdown of proper complement regulatory mechanisms leads to pathological conditions in which complement effectors cause chronic inflammation and damage to host cells. Despite many efforts, only one complement-targeted therapeutic exists in the clinic. Compstatin peptides are a family of low molecular mass complement inhibitors that block cleavage of complement component C3 and downstream effector functions of complement activation. Over recent years, the compstatin sequence has been iteratively optimized to improve binding and complement inhibitory activity (Gorham et al., Exp. Eye Res., 2013). Hydrophobic interactions are critical for C3 binding, however they also promote peptide aggregation in aqueous solution. We have designed several new compstatin peptides containing non-natural amino acid modifications and polar N-terminal amino acid extensions. Presence of N-terminal extensions alone, as well as incorporation of alpha-modified alanine analogs, maintained similar activity to current top compstatin peptides, as measured by in vitro and cell-based assays, and markedly improved solubility as measured by UV absorbance and RP-HPLC experiments. These peptides are candidates for development of therapeutics for a wide variety of complement-mediated diseases.

 Abstract Title: Virtual Screening Based Drug Discovery for Complement-Mediated Inflammation

Abstract: In the complement system, there is balance between positive and negative feedback loops that regulate innate immunity. While feed-forward loops promote complement activation in the presence of foreign surfaces, negative feedback processes are responsible for regulating complement activation on host cells and tissues. Perturbations to this system, such as chronic overstimulation by C5a or C3a anaphylatoxin, are associated with various autoimmune pathologies. In healthy individuals, C5a is a key complement protein that induces inflammation and promotes chemotaxis of circulating leukocytes. Overexpression of C5a is linked to autoimmune disorders such as inflammatory bowel disease and lupus. In search of an inhibitor of the interaction of C5a with the C5a receptor, a peptide named PMX53 was designed [1] that has beta turn structure similar to the C-terminus of C5a [2]. Despite potent inhibitory activity, PMX53 exhibits poor bioavailability in vivo. In an effort to find an alternative antagonist of C5a, we conducted high throughput virtual screening for compounds with predicted structural characteristics similar to PMX53. We used pharmacophore screening to identify chemical compounds with similar topological arrangement of physicochemical properties as in PMX53. Pharmacophore hits were docked to C5aR, and molecules were ranked using predicted binding energies and cheminformatic tools, focusing on compounds that interact with the C5a receptor at loci similar to PMX53. Data compiled from docking calculations, root-mean-square distance clustering around the site of docking on the C5a receptor, and predictions of chemical characteristics relevant to Lipinski’s rules enabled selection of twenty promising compounds. Future studies will characterize these top hits experimentally with procedures including binding and biochemical assays.

[1] Klos A, et al (2013) Pharmacological Reviews 65:500-543.

[2] Zhang L, et al (2008) Biopolymers: Peptide Science 90:803–815.

 

Abstract Title: Low fluence thresholds for in vitro photodynamic therapy

Abstract: Photodynamic therapy (PDT) is a type of medical treatment which uses light and specialized photosensitive drugs that convert the light into useful chemical energy (e.g. reactive oxygen species). In the research setting PDT has been shown effective against a variety of diseases including numerous cancers, skin diseases, macular degeneration, and many others. Despite these successes, translation to the clinical setting has been mostly limited to the treatment of superficial diseases. This is largely due to the difficulties associated with delivering light to deep tissues using traditional light sources (lasers and LEDs). A variety of methods have been proposed to overcome this by delivering light macroscopically to deep lesions with nanoscintillators, long-lived phosphorescent nanoparticles, and radionuclides for Cerenkov luminescence among others. With respect to traditional clinical light sources, these methods produce light at lower and more effective wavelengths, but at much lower intensities. Our work intends to characterize and quantify the effectiveness of PDT under these unique conditions to determine minimum light thresholds to aim for when developing novel light sources. Experiments were conducted in white, clear-bottom 96-well plates on a variety of cancer cell lines including brain, breast, and kidney. Cells were treated with light using custom built overhead LED illumination arrays at varying intensities and assayed for viability using the WST-1 proliferation assay. Using an FDA approved photosensitizer (aminolevulinic acid), our results show that blue light is approximately ten times more effective for all cell lines when compared to clinically used red light. By using a more efficient photosensitizer (TPPS2a) the amount of light needed can be decreased by another order of magnitude, thereby reducing the minimum light dose needed to see an effect from PDT to tens of mJ/cm^2 and approximately 100 times less than has been reported in previous literature.

 

Abstract Title: IDENTIFYING THE OPTIMAL ALGINATE HYDROGEL AND TRANSPLANT SITE FOR ALGINATE MICROCAPSULE TRANSPLANTATION

Abstract: Islet microencapsulation, where the islets are coated with a biocompatible polymer, has demonstrated various degrees of success in small and large animal trials. This success depends on many factors including alginate purity and the location at which the devices are transplanted. In order to address these issues, we evaluated alginate microcapsules made from two types of alginate hydrogels transplanted to one of three transplant sites into athymic nude mice to characterize changes in microcapsule morphology and foreign body and vascular response post-transplant. Microcapsules generated with an air-driven, electrostatic microcapsule generator using either Sigma or UP LVM (Ultra-Pure Low Viscosity Mannuronate) alginate solution at two concentrations (1.5% & 3%) were crosslinked in a gelling solution containing a 120mM calcium chloride solution. The microcapsules were characterized using an inverted microscope after which they were transplanted at one of three sites in C57Bl/6 mice – subcutaneous, intraperitoneal and inside the epididymal fat pad. The recipients were monitored for a 2 week period after which they were euthanized and the microcapsules were carefully collected for histological analysis. The results of this study showed that the subcutaneous and intraperitoneal transplant sites demonstrated greater foreign body and collagen deposition than the epididymal fat pad site (see fig). Microcapsules made from Sigma alginate were also found to be less biocompatible than those made with UP LVM alginate as evidenced by a greater tissue reaction surrounding the former. The results of this study suggest that the epididymal fat pad site may be an immune privileged site for islet and encapsulated islet transplantation. In vivo studies can also compare and evaluate biomaterial biocompatibility enabling identification of the optimal alginate concentration and purity for use in encapsulated islet and stem cell transplantation to improve graft success.

 

Abstract Title: Simultaneous Cryogenic Anchoring and Radiofrequency Ablation for Cardiac Arrhythmia Treatment

Abstract: Cardiac arrhythmia is a disease characterized by abnormal electrical conduction in the heart that results in ineffective pumping. Dysfunctional nodes in the conduction pathway or in the cardiac muscles lead to irregular heartbeat patterns that can potentially induce severe complications such as cardiac arrest. Radiofrequency ablation, the current gold-standard cardiac arrhythmia treatment procedure, has proven effective but suffers from shortcomings due to instability between the RF tip and the target site. We tested a cryogenic anchoring system that can be combined with RF ablation to create a stable ablation tip that physically attaches to the cardiac surface. A prototype anchor that incorporates RF ablation with a cryogenic adhesion system was shown to anchor to cardiac tissue with enough adhesion strength to maintain attachment during cardiac contractions. The thermal interference between heat generated by RF ablation and the cryogenic temperatures of cryogenic anchoring was not significant, demonstrating the feasibility of utilizing both technologies simultaneously. These results suggest that combining RF ablation and a cryogenic anchor into one catheter enables a physician to treat cardiac arrhythmia with improved stability and may be utilized for anchored tissue ablation of other organs where instability is an issue.
 

Abstract Title: Simultaneous Cryogenic Anchoring and Radiofrequency Ablation for Cardiac Arrhythmia Treatment

Abstract: Cardiac arrhythmia is a disease characterized by abnormal electrical conduction in the heart that results in ineffective pumping. Dysfunctional nodes in the conduction pathway or in the cardiac muscles lead to irregular heartbeat patterns that can potentially induce severe complications such as cardiac arrest. Radiofrequency ablation, the current gold-standard cardiac arrhythmia treatment procedure, has proven effective but suffers from shortcomings due to instability between the RF tip and the target site. We tested a cryogenic anchoring system that can be combined with RF ablation to create a stable ablation tip that physically attaches to the cardiac surface. A prototype anchor that incorporates RF ablation with a cryogenic adhesion system was shown to anchor to cardiac tissue with enough adhesion strength to maintain attachment during cardiac contractions. The thermal interference between heat generated by RF ablation and the cryogenic temperatures of cryogenic anchoring was not significant, demonstrating the feasibility of utilizing both technologies simultaneously. These results suggest that combining RF ablation and a cryogenic anchor into one catheter enables a physician to treat cardiac arrhythmia with improved stability and may be utilized for anchored tissue ablation of other organs where instability is an issue.

 Abstract Title: Development of Combinatorial, Biomaterial-Mediated Gene Therapies for Spinal Cord Tissue Regeneration

Abstract: The local environment after spinal cord injury (SCI) lacks cues to support axon growth, cell survival and remyelination and in fact inhibits these processes. Development of clinically effective strategies to restore function after SCI requires consideration of multiple aspects of this inhibitory environment. We aim to develop a multifaceted gene therapy for SCI repair that delivers vectors encoding for cues that enhance cell survival, reduce inflammation and promote axonal growth and remyelination. For these studies, biomaterials fabricated from poly(lactide-co-glycolide) (PLG) were used as a platform for tandem, localized delivery of lentiviral vectors encoding regenerative factors, including sonic hedgehog (SHH), neurotrophin-3 (NT-3) and interleukin-10. Delivery of each individual vector in a mouse model of SCI resulted in improved repair. In addition, the combination of SHH and NT-3 vectors resulted in significant increases in the number of regenerated axons through the bridge platforms and myelination of these axons. Although PLG biomaterials are useful as an investigational platform for identifying clinically relevant gene therapy targets in vivo, they require that sections of spinal cord be removed prior to implantation and do not adequately resemble the native tissue. As a clinically viable alternative, we are also investigating the use of hydrogel biomaterials based on hyaluronic acid, which is abundant in the extracellular matrix of the uninjured spinal cord and down-regulates the inflammatory response after injury for their ability to mediate SCI repair.

 

Abstract Title: OPTIMIZATION OF ENCAPSULATION PARAMETERS TO ACHIEVE IDEAL ALGINATE MICROCAPSULE MORPHOLOGY

Abstract: Human islet allotransplantation has shown moderate long term success in a small subset of type I diabetics, albeit with the need for chronic immunosuppression. Alginate microencapsulation, where the islets are coated with a biocompatible polymer, has demonstrated successful reversal of diabetes without the need for immunosuppression. Alginate microcapsules of uniform size (~300µm) and shape have been postulated to possess ideal morphological parameters to allow for optimal oxygen and nutrient diffusion without exposing the interior of the encapsulated islet to hypoxic stress. In this study, we have identified two variables i.e. alginate concentration and the electrostatic potential of the encapsulators which impact microcapsule size. We aimed to study the effect of varying these parameters in order to achieve alginate microcapsules of a consistent size ((~300µm) and shape. Alginate hydrogels were made using Sigma Alginate at concentrations of 1.5% or 3%, and converted into microcapsules using an air-driven electrostatic microcapsule generator and polymerized in a gelling solution composed of 120mM Calcium Chloride solution after which their morphology was assessed. The results demonstrated that the mean diameter increased with increasing alginate concentration (394.1±2.0µm, 1.5%; 492.9±0.7µm, 3%, p=0.001). Alginate microcapsules made using 2.1% Sigma alginate were gelled at increasing voltages from 4kV to 10kV and their mean diameter and roundedness were evaluated (See Fig). In this study we observed that while 6kV yielded microcapsules with a diameter of 520.5±46.8µm, at 8kV, the microcapsules generated were of greater uniformity, though slightly larger (581.4±11.8µm). Future studies will evaluate the effect of varying other parameters, i.e. needle bore, air pressure and temperature, concentration of gelling solution and duration of crosslinking on microcapsule morphology to achieve consistent and uniform microcapsules for the purpose of islet transplantation

 Diagnostics and Imaging

 

Abstract Title: In vitro selection of structure-switching aptamers for insulin and the design of a continuous, in vivo insulin-monitoring device

Abstract: Diabetes mellitus affects more than 26 million Americans nationwide. More cases of kidney failure, limb amputations, and blindness are associated with diabetes than any other illness. The disease is caused by an individual’s inability to produce or utilize insulin. Some of these individuals rely on daily insulin injections to maintain their blood glucose levels (BGL). Current methodology for determining insulin dosage is based solely on BGL measurements. This approach is inadequate, however, because it fails to take into account how much insulin is circulating in the blood and results in poor glycemic control. Additionally, insulin detection is necessary as a feedback and safety mechanism for insulin pumps and artificial pancreas systems. The lack of an integrated insulin sensor in these devices puts a user at risk for coma or death from hypoglycemia because there is no sign of malfunction until BGL drops to dangerous levels. It is imperative that an insulin detection system be developed that can quickly and accurately assess a person’s circulating insulin levels outside of a clinical setting. To this end, we are developing an aptamer-based insulin sensor for continuous, in vivo detection. Aptamers are short nucleic acid molecules that are selected from a large random sequence pool to bind their targets with high specificity by iterative rounds of selective amplification. I report our selection strategy and progress for a structure-switching insulin aptamer. We have developed a sensor design that can take advantage of this structure-switching ability as a means for real-time monitoring of insulin in a point-of-care device.

 

Abstract Title: Characterizing Wicking Under Non-ideal Conditions in Paper Analytical Devices

Abstract: Paper analytical devices are emerging technologies with the potential to revolutionize point-of-care diagnostics. Paper is a low cost, ubiquitous, and self-wicking material, thus is an ideal substrate for developing simple-to-use, portable, and disposable devices for fluid specimen analysis. Existing devices are typically simple lateral flow tests whose functions are limited to low level qualitative detection of analytes. To significantly expand functionality of paper analytical devices, it is necessary to better understand fluid transport within the channel networks defined on the paper substrate. In this study, we investigated fluid wicking in channels that were created by printing and melting a wax-based ink on a sheet of filter paper. Wet-out flows through porous media are often described using the Lucas-Washburn equation, which models the porous medium as a bundle of rigid capillaries. However, this classical model is insufficient in describing fluid retardation effects such as of swelling, channel boundary, and evaporation, all of which are evident in operations of most paper devices. We experimentally investigated these retardation parameters and developed a modified version of the Lucas-Washburn equation which provides a more accurate prediction of fluid wicking through paper channels under non-ideal conditions. In addition to constant width channel wicking, we analyzed the effect on overall wicking due to a variety of channel geometries that may be present in complex fluidic circuitry. Collectively, these results are expected to help develop more functional paper analytical devices.

 

Abstract Title: A Novel Microfluidics Device for Determination of the Protein Interaction Affinity, Kd, by Quantitative FRET-based assay

Abstract: The protein interaction dissociation constant, Kd, is a fundamental parameter for characterization of the affinity for protein-protein interactions, or ligand-receptor interactions. There are tremendous efforts in developing various technologies to determine the Kd, such as surface plasma resonance (SPR) and isothermal titration calorimetry (ITC). However, these technologies either need surface conjugation or large amounts of proteins, and therefore they are not suitable for large scale, high-throughput assays. Thus, we have developed a microfluidics device, with a chip constructed from glass wafers, and a polydimethylsiloxane (PDMS) membrane, to perform Forster Resonance Energy Transfer (FRET) assays with the goal of ascertaining quantitative FRET measurements in order to determine Kd. FRET occurs due to energy transfer from an excited donor fluorophore to an acceptor fluorophore when their respective emission and excitation spectra overlap more than 20%, and their distance is between 1-10nm. A Labview computer interface controls the dilution and mixing of two protein solutions, which is then imaged by a Leica SP5 confocal microscope’s PMT’s. The FRET images are processed using a program written in Matlab code, which contains a quantitative FRET measurement equation developed at the Liao lab for determining true FRET emission, and subsequently Kd. Here, we present our concepts, fabrication of the device, and testing done towards obtaining Kd from quantitative FRET determination.

Abstract Title: High Throughput Cell Viability Assay Using Deformability Cytometry

Abstract: Cell viability assays are a critical component of many drug sensitization studies yet many still rely on bulk analysis on representative samples to form conclusions on the population. Deformability cytometry has the ability to bring large population analysis to eliminate bias that can result from bulk analysis. In this study we evaluate the ability to detect live and dead cells in a population without the need for staining or labeling. Deformability cytometry uses the deformation of cells under high, controlled shear to pick up on the differences between the mechanical properties of live and dead cells. Chromatin modifying drugs were used on Jurkat cells, a T-cell leukemia cell line, in order to induce a controlled, dose dependent cellular apoptosis. The cells were analyzed using deformability cytometry for three continuous days, along with a live/dead staining assay as a gold standard to determine population viability. Using statistical algorithms, the cells could be clustered into two distinct populations (live and dead) using only the deformability (a measure of cell strain) and size of the cells. The algorithm allowed for accurate measures of viability for one of the dosages, but gave a large error for a steeper dosage. These results show that our device has potential to function as a high throughput viability assay that is much cheaper, faster, and reliable than conventional tools.

Abstract Title: Improving assay performance with an acoustic-driven microfluidic platform

Abstract: The development of an integrated immunoassay platform is crucial for providing diagnostic tools for global health applications. With the emergence of microfluidics, there is an increased focus in addressing this need. While many platforms have been created to successfully complete an immunoassay, they still require bulky external equipment to manipulate fluid within the device. Using technology developed in the BioMiNT lab, we’ve created an immunoassay platform that is capable of on-chip pumping and mixing with lateral and vertical-cavity acoustic transducer (LCAT & VCAT respectively) coupled to a piezoelectric transducer (PZT). Using standard photolithography methods, a dual-layer SU-8 master is fabricated so that devices can be casted with PDMS. The devices are then contact-bonded to glass slides adhered with antigen-spotted nitrocellulose pads. Once they are primed with blocking buffer to introduce air bubbles, the devices are actuated with a PZT to drive fluid pumping and mixing. The reagents are pumped into the device serially and after completion, the spotted antigens on the pad become a dark purple hue, indicating an antibody-antigen binding event. For quantitative results, the intensities are evaluated using computer software. Using the LCAT/VCAT platform, an immunoassay can be performed in a fifth of the time needed for standard protocols and with increased signal intensity. Future work will involve expanding the device to allow for an automated point-of-care platform.

Abstract Title: Screening cell mechanotype using a parallel filtration assay

Abstract: Cell mechanical phenotype or ‘mechanotype’ is emerging as a valuable label-free biomarker. For example, marked changes in the mechanical properties of cells occur during malignant transformation and in response to chemotherapy treatment. However, a simple and scalable technique to measure cell mechanotype is lacking. Here we describe a parallel filtration assay, which enables the mechanotype of multiple samples to be simultaneously measured by driving cell suspensions through porous membranes using uniform air pressure. We establish the effects of cell density and driving pressure on filtration so that a measurable fraction of samples transit through membranes with a particular pore size; the fraction of sample retained above the membrane relates to the deformability of cell samples. Using this method, we measure variations of cell mechanotype induced by treating cells with cytoskeletal-perturbing and chemotherapeutical drugs. To further validate the method, we probe human and mouse ovarian carcinoma cells, revealing that both mesenchymal and drug-resistant ovarian cancer cells are more deformable than epithelial and drug-sensitive cells. Our results demonstrate a method to screen cell mechanotype that has potential for broader clinical application.

 Abstract Title: Improved Fertility Monitoring

Abstract: Millions of couples worldwide have difficulties trying to conceive. These couples are not necessarily clinically infertile, but instead have difficulty timing intercourse to achieve pregnancy. Failure to conceive can lead to serious psychological distress and lowered life satisfaction. Since the menstrual cycle fluctuates monthly, women use fertility monitoring devices to identify ovulation. This is crucial, because women are only fertile within a three-day window prior to ovulation. Current fertility monitoring devices on the market do not define the fertile window early enough or accurately enough to effectively help couples time intercourse. In fact, popular basal body temperature devices actually identify the ovulation one day after it occurs. Our design team is developing the Couple’s Fertility Monitor to help couples and clinicians overcome the limitations of existing devices. Utilizing a novel sympto-thermal method and proprietary algorithms, our device will predict ovulation within a 24 hour window 5-7 days in advance. Thus, the Couple’s Fertility Monitor seeks to revolutionize the fertility monitoring device industry by achieving unprecedented ovulation prediction, assisting with male infertility, and helping couples time intercourse to conceive.

 

Abstract Title: High-Resolution, Depth-Resolved Imaging of Microglia and Microvasculature in Mouse Brain Using Optical Histology

Abstract: Introduction: Alzheimer’s disease is an irreversible neurodegenerative disease that impacts around five million Americans. The complexity of the brain makes it difficult to determine the exact cause of Alzheimer’s, thus inhibiting progress towards developing an effective treatment. Recent research has shown that the brain’s microvasculature and microglia, immune cell for the central nervous system, may be involved in the development of Alzheimer’s disease. Using “optical histology,” a method developed in our lab that combines chemical and optical techniques, we are able to image the microvasculature and microglia in a mouse brain at high-resolution.

Materials and Methods: To perform optical histology, we perfused DiI, a lipophilic dye, through the left ventricle of a euthanized transgenic mouse with GFP-labeled microglia. DiI stains the endothelium, subsequently labeling the systemic vasculature and microvasculature. The brain is then carefully extracted and a 500 micron-think brain section is incubated in the optical clearing agent, FocusClear for 3 hours. The brain is imaged using confocal microscopy with a 488 nm laser to excite DiI and GFP fluorescence. Acquired images are processed through MATLAB and presented as a maximum intensity projection map, which is a 1D image comprised of the maximum intensity pixel value at each pixel location over all the slices in the z-stack.Results and Discussion: Using this method, we were able to acquire micron-resolution images of the cerebral microvasculature and microglia with a wide field-of-view.Conclusion: We successfully demonstrated our ability to use this method to acquire high resolution images of microvasculature and microglia in a mouse brain. The optical clearing component of optical histology enables visualization of fluorescent structures to a depth of 500 microns into the tissue. Optical histology can potentially be useful in studying the behavior of microglia and microvasculature in Alzheimer’s disease.

Abstract Title: A Microfluidic Device for Dissociating Tumor Tissue into Single Cells

Abstract: The ability to extract molecular information form solid tumor specimens could revolutionize patient care by drastically improving diagnosis/prognosis and enabling personalized medicine. Currently dissociation procedures involve harsh or lengthy enzymatic digestions that can compromise sample quality. A more powerful strategy would be to immediately process the tumor tissue specimen and either analyze or fix the single cell suspension at the patient’s bedside. Here we seek to achieve this goal by developing a novel microfluidic device. Our device design includes branching channels that contain constriction and expansion regions. Our design principle is to gradually reduce cross-section through a series of bifurcating stages. The constriction and expansion regions induce flow disturbances that help mix the sample and generate fluidic jets at different length scales to generate shear forces necessary to dissociate cell aggregates. The performance of our device was characterized in terms of cell recovery and surface biomarker expression levels. Spheroids dissociated using standard treatment with trypsin-EDTA and mechanical shearing were used as control samples. Using only the device to dissociate HCT116 spheroids, we have observed comparable single cell recoveries to the trypsin control. However, if the device is employed after a brief trypsin treatment, we observe a 3-fold increase in cell recovery yield. Biomarker preservation is demonstrated by assessing biomarkers that are known to be trypsin insensitive. In summary, we have designed and tested a microfluidic dissociation device and found significant improvement in tumor tissue dissociation relative to enzymatic digestion. We have also achieved results under enzyme-free conditions that are comparable to or exceed trypsin digestion, thus preserving biomarker expression level for molecular diagnostics.

Abstract Title: Super-Resolution Imaging of Focal Adhesion Proteins on Nanopatterned Surfaces

Abstract: Cells are sensitive to their environment especially to the extracellular matrix including the surface they interact with. Particularly, cell surface proteins involved in cell-to-substrate interactions are nanometers in size and thus changes to the surface on the nanoscale affect cell activity. Past studies have shown that on nanolined surfaces cells (bacterial and mammalian) adhere and migrate parallel to the gratings, while they barely adhere on pillared surfaces. Currently, most microscopy techniques that observe internal cell structures that are a few nanometers in size are conducted by fixing the cells and imaging them at high resolution or by destroying them for biochemical analysis. In addition, these imaging set-ups are often complex and cannot capture protein organization below the diffraction limit of light and their dynamics that occur within the order of milli-seconds. Emerging technologies from light microscopy not only have the potential to provide super-resolution images but to also obtain information about spatio-temporal dynamics of proteins in living cells. Here, we use a super-resolution imaging technique that uses a phasor representation to obtain a super-resolution z-stack images of protein structures in live cells. In combination with confocal microscopy, we are able to take 30-50 nm z-stacks of cells which are grown on substrates of different nanotopographies (flat and lined) to reconstruct a 3D nanometer image of the focal adhesion structures formed at cellular protrustions. By fluorescently tagging individual proteins involved in the formation of adhesion points, we have been able to analyze the organization of the proteins on nanoimprinted surfaces in order to study how the surfaces affect cell adhesion and mobility. From these studies, we have a better understanding of how modified surfaces can optimize cell attachment and detachment for biomedical implants and anti-bacterial surfaces.

Abstract Title: Effect of ICG Concentration on the Fluorescence Emission Characteristics of Erythrocyte-Mimicking Optical Nanoprobes

Abstract: Indocyanine green (ICG) is an FDA approved near-infrared (NIR) fluorophore that has been used in choroidal angiography, measurement of cardiac output, and assessment of hepatic function. Rapid clearance of ICG from the vasculature (half-life ˜ 3-5 min) limits the dye’s usefulness to short times following administration. We aim to deliver ICG in an appropriate formulation that can be utilized for optical imaging and phototherapeutic applications over long periods of time.

We have engineered optical probes derived from hemoglobin-depleted erythrocytes doped with ICG. We refer to them as NIR erythrocyte-mimicking transducers (NETs). To utilize NETs as effective probes for fluorescence imaging, we must ensure that they provide maximal emission. Here, we aim to maximize the fluorescence quantum yield of NETs by changing the concentration of ICG loading over a broad range (5-100 µM).NETs are fabricated by hypotonic treatment of erythrocytes (80 mOsm, pH = 8, 20 min, 4°C), followed by incubation with various concentrations of ICG in loading buffer (160 mOsm, pH = 8, 5 min, 4°C). Using the absorption and fluorescent spectra of the NETs, we can determine the relative fluorescence quantum yield of the NETs at a given ICG concentration.Low concentrations of ICG result in weak fluorescence emissions, while higher concentrations quench the fluorescence signal. Therefore, NETs provide maximal fluorescence when an optimum concentration of ICG is encapsulated.

 

Abstract Title: Activatable FRET Nanosensor for Visualizing MT1-MMP Activity in Single Cancer Cells

Abstract: Membrane type 1 metalloproteinase (MT1-MMP) is an important marker for tumor malignancy since it can directly degrade extracellular matrix and promote cancer cell metastasis. Detecting MT1-MMP activity of cancer cells is an important index to monitor tumor malignancy in cancer diagnosis. However, there is lack of a simple method which can visualize MT1-MMP activity of a cancer cell with high resolution. Here, a MT1-MMP activatable quantum-dot (QD) nanosensor was engineered to visualize the MT1-MMP activity at the single cell level. The QD-based Förster resonance energy transfer (FRET) nanosensor was cleaved by pericellular MT1-MMP of cancer cells, which resulted in a marked change of donor (QD)/acceptor (Cy3) emission ratio and activated cell penetration sequences allowing uptake by the cell. By Cellular FRET images and quantified intracellularly accumulated nanosensor, our nanosensor is able to differentiate various levels of MT1-MMP activity in cancer cells. Thus our enzyme activatable peptide/QD-based FRET nanosensor can visualize the enzyme activity of a cancer cell, providing a simple method for monitoring tumor malignancy

Abstract Title: ON-CHIP CELL MOTILITY ANALYSIS USING LENSFREE HOLOGRAHIC MICROSCOPY

Abstract: Cell motility is a vital process that is important both in development and homeostasis of organisms. Quantitative studies of cell motility are necessary for understanding these processes and for drug discovery. Such studies are typically performed using optical microscopes with attached environmental chambers to image migrating cells. However, these instruments are costly and bulky and limited in throughput by the size of the field-of-view. We present a compact cell monitoring platform utilizing a wide-field (24mm2) lensless holographic microscope that enables high-throughput automated tracking of single-cell motion and can be used in standard laboratory incubators. Our lensfree on-chip imaging platform works by recording the interference patterns of the light scattered from the target objects (e.g. cells) with the unscattered background light, using a CMOS imager chip. Image analysis software is used to detect cells and record their positions. Previously, lens-free imaging has successfully been used to statically image particles and cells, automated cell counts, tracing trajectories of sperm, and evaluating water quality. Here, we employ lens-free imaging to track the motions of mammalian cells for a full day in an automated manner. To demonstrate our platform’s cell

Abstract Title: 3D Tissue Reconstruction by Using Deep Tissue Fluorescence Imaging System and FLIM

Abstract: We have demonstrated a new detection method used in two-photon fluorescence microscope called the DIVER (Deep Imaging via Enhanced-Photon Recovery) with enhanced capabilities for deep tissue imaging. The microscope uses a large area photo-detector to collect scattered emission photons directly from the wide area of a specimen. This detection scheme allows for increasing the imaging depth in tissues by about 6 folds due to a unique detector design and its increased photons collection efficiency compared to conventional methods. The DIVER system is also capable of performing Fluorescence Lifetime Imaging (FLIM), a very powerful tool to segregate various features in cells and tissues. The combination of deep tissue imaging and fluorescence lifetime provides contrasted images based on physiological parameters at depths that are not achievable by conventional microscopes. The DIVER potentially can be useful for imaging of various tissue samples, in particular for human skin cancer diagnostics, providing 3D cellular resolved images. We present the principle of operation of the microscope and show images obtained in unstained tissues in the heart, liver, kidney, lungs and other organs of mice. As an example of SHG in biological tissue, we have imaged the medial thigh muscle of a mouse obtained from the upper region of the leg. The muscle, roughly 3-4 mm thick, multiple z-stack images were acquired at field of view ranging from 400-900 ?m, we can see the organization of the sarcomeres as well as the striations present in each sarcomere. We also have taken z-stack of the mouse heart where upon 3D reconstruction we can differentiate the different layers of the heart muscle as well as the ventricle.

Abstract Title: An Automated and Compact Microfluidic Device for Rapid Concentration of PET tracers

Abstract: Position emission tomography (PET) has become a critical tool for in vitro and in vivo cancer biology research leading to more accurate diagnostics and post treatment analysis. The imminent shift from centralized PET tracer synthesis facilities to decentralized production at imaging sites has increased the need for miniaturized, simple to use post synthesis machinery for probe purification, formulation, concentration, and quality control. While high performance liquid chromatography (HPLC) systems are the gold standard for purification, a major drawback is the added sample volume during HPLC purification. This dilution can hinder the ability to inject sufficient amount of tracer to obtain a high-quality image, especially in studies using small animals such as rats or mice, where maximum injection volumes are very limited. As such, a dilute tracer sample therefore requires concentration prior to injection. An ideal concentration system would be compact to minimize the amount of radiation shielding needed and would provide fast evaporation rates. In our previous work, we have demonstrated the ability to rapidly concentrate PET tracers on a microfluidic device through means of membrane distillation. The device demonstrated evaporation rates and a reduced physical footprint compared to commercial rotary evaporators typically used for concentration. In this current study, we plan on improving the previous design allowing for even faster evaporation rates, a wider range of chemical and PET tracer compatibility, full device automation, and direct integration into commercially available synthesis and purification systems.

 

Abstract Title: Microflotronic Pulse Measurement: A Transparent, Flexible Pressure Sensing Platform for Continuous Noninvasive Arterial Tonometry

Abstract: Cardiovascular disease inflicts hundreds of billions of dollars of financial burden and immeasurable human suffering worldwide. Early diagnosis and treatment of cardiovascular disease is crucial to prevent mortality and improve quality of life. However, traditional annual measurements of peripheral blood pressure have many limitations in the diagnosis of cardiovascular disease states. Arterial tonometry measures blood pressure continuously and noninvasively by pressing a pressure transducer over the target artery to reproduce the entire blood pressure waveform to facilitate pressure curve analysis to diagnose cardiovascular disease states. By applying a combination of emerging microfluidic and electronic technologies, a miniature, flexible, transparent, highly sensitive and wearable pressure sensor has been implemented, referred to as a microflotronic sensor. The sensor exhibits a fast response time on the order of tens of milliseconds and a high sensitivity of 0.1kPa-1. This sensitivity is among the highest in impedance-based flexible pressure sensors. When implemented in a sensor matrix configuration, the transparent device can be easily aligned over the target artery to perform arterial tonometry. In addition, the thin ultraflexible (270µm) polymer substrate of the microflotronic sensor can be worn comfortably for extended periods of time. Importantly, the diagnostic value of the microflotronic device was successfully demonstrated by accurately reproducing the waveforms of the radial and carotid arteries in order to subsequently compute the hemodynamic parameters of mean arterial pressure, central systolic pressure, and carotid-radial pulse wave velocity.

Abstract Title: Fructation and Cross-linking of Collagen Hydrogel

Abstract: Fructation is a significant issue because it has applications to health improvement and age wear reduction. Also, it can be used to increase durability of biomaterials through the introduction of rigid rings into the final biomaterial structure. D-Fructose and proteins undergo fructation (glycation by fructose) through the process of a non-enzymatic reaction to create permanently cross-linked fluorescent derivatives termed advanced glycation end-products (AGEs). This modification takes place through a Maillard Reaction and works differently on aldoses (sugar molecule with an aldehyde substituent) compared to ketoses (sugar molecule with a ketone substituent). A Maillard Reaction with D-fructose as the reducing sugar gives Heyn’s products as opposed to the Amadori products seen in the Maillard Reaction of D-glucose. While a significant amount of research has been done on the D-glucose driven cross-linking, there has been little research looking at D-Fructose and its products.
In order to test the cross-linking effects of fructose, a collagen hydrogel was synthesized. On top of this hydrogel, a layer of fructose solution was added. Over the course of 30 days, one-photon fluorescence spectroscopy and in situ two-photon optical microscopy imaging and spectroscopy was used to detect the fluorescent structures and determine in what ways fructation had altered their forms. The two-photon fluorescence (TPF) emission was centered at about 460 nm for 730 nm excitation wavelength and shifted to 480 nm when we changed the excitation wavelength to 790 nm. The one-photon fluorescence emission was centered at about 416 nm when excitation was 330 nm. It red shifted and split into two peaks centered at about 430 nm and 460 nm for 370 nm excitation; 460 nm peak became predominant for 385 nm excitation and further shifted to 470 nm for 390 nm excitation. Second Harmonic Generation (SHG) and TPF imaging showed restructuring of hydrogels upon this modification.

Abstract Title: Controllable microfluidic synthesis of high ionic solution encapsulated liposomes for diagnostic applications

Abstract: In recent years, liposomes have been attracted much attention in the fields of cancer therapeutic and drug delivery. Besides, liposomes can also be applied as a sensor component for biochemical detection. Bashir group reported ELISA-inspired approach for biological detection based on liposome tagging and ion-release impedance spectroscopy. However, liposomes fabricated with current methods have polydispersed populations, limited shelf life, and varying degrees of mutilamellarity. Encapsulation efficiency is particularly poor in conventional liposome formation work. In this work, we present a microfluidic platform for continuous generation of stable and monodispersed lipid vesicles. Our approach utilizes a microfluidic flow-focusing device to generate vesicles with a narrow size distribution by precisely controlling the system’s fluid flow pressure. The formation of lipid bilayer of vesicles is followed by a solvent extraction process. The droplets generated by this method are quite stable with the 3-month shelf life at least. Liposomes tagging and encapsulating high ionic solutions can be used as the sensing element by baking down the lipid and releasing ionic. We individually test 10XPBS-encapsulated vesicles with DSPC and DPPC lipid layers These liposomes are stable in deionized water but permeabilized for ionic release upon heating. In this study, these vesicles are characterized and fluorescein isothiocyanate-dextran is also encapsulated to check the leakage and stability issue. The method we developed can generate stable vesicles with monodispersed size and has high potential to apply in sensor fields.

 Abstract Title: FRET visualizes EphA4 activity at different compartments or the plasma membrane

Abstract: EhpA4 belongs to the family of Eph receptors and is well known for its important role in axon growth cone guidance event during development stage. However, how EphA4 activity is regulated in space and time remains to be elucidated in these neural cells. Here, we generated an EphA4 biosensor based on fluorescence resonance energy transfer (FRET), which enables the visualization of EphA4 activity with high spatiotemporal resolution in live cells.
I engineered the EphA4 biosensor by inserting an EphA4-specific substrate peptide and a SH2 domain in between an ECFP (FRET donor) and an YPet (FRET acceptor) fluorescent protein. Upon ephrin activation, the tyrosine in substrate sequence would be specifically phosphorylated, and the phosphorylated tyrosine would bind to the SH2 domain. This binding would cause a conformational change of the biosensor, which would lead to a FRET signal change. Both in vitro and in cell methods were applied to characterize the efficiency and specificity. Emission spectrum was measured in in vitro assay by spectroscopy.
I successfully generated the cytosolic, lipid-raft targeted and non lipid-raft targeted EphA4 biosensor by molecular cloning. I found that non lipid-raft targeted EphA4 biosensor responds to ephrinA1 and ephrinA3 stimulation faster and stronger than lipid-raft targeted one, while destroying cytoskeleton with cytoD reverses the response. Further investigation will be performed to understand the underlying mechanism.

Abstract Title: Non – invasive optical metabolic imaging of three-dimensional cardiac tissue model in a polydimethylsiloxane microfluidic device

Abstract: There is a growing importance of development of microfludic device based, microphysiological tissue systems which simulates structure and function of human organs. Such tissue chips find application in drug toxicity screening, point-of-care diagnostic devices, targeted drug delivery and so on. Optical imaging proves to be a powerful, non- invasive tool to monitor development of such tissue systems. In this work, we show label-free metabolic imaging of induced pluripotent stem cell derived cardiomyocyte (iPS-CM) 3D spheroids co-cultured with endothelial cells (to form vessel network) and fibroblast in tissue microchamber of a polydimethylsiloxane (PDMS) microfluidic device. We performed fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorophore, reduced nicotinamide adenine dinucleotide (NADH), a metabolic coenzyme which plays myriad of roles in cellular oxidation and reduction reactions. The phasor approach to FLIM was applied to map free to protein bound NADH ratio distribution in the iPS-CM spheroid and surrounding vessel network, indicative of the metabolic status of the biological system within the microtissue chamber. This technique was also employed study the metabolic response of the system to application of drugs. FLIM of NADH was performed after administration of potassium cyanide (KCN) which is a reducing agent. Phasor analysis of the NADH lifetime distribution showed an increase in free to bound NADH ratio with time, after adding the drug. This reflects a shift of metabolic state from oxidative phosphorylation to glycolysis which corresponds to the fact that KCN blocks cellular respiration causing an increase in free NADH. Hence, we show mapping of metabolic activity and non-invasive monitoring of drug response of a microphysiological tissue system in a PDMS microfluidic device chamber employing a non-destructive, label-free, optical microscopy technique. Work supported in part by NIH grants P50GM076516, P41GM103540 and UH2TR000481-0.

 

Abstract Title: Real-time intracoronary imaging of atherosclerotic plaques by the back-to-back integrated optical coherence tomography (OCT)-intravascular ultrasound (IVUS) probe

Abstract: We have developed a novel integrated optical coherence tomography (OCT)-intravascular ultrasound (IVUS) probe, with a 1.5 mm-long rigid-part and 0.9 mm outer diameter, for real-time intracoronary imaging of atherosclerotic plaques and guiding interventional procedures. By placing the OCT ball lens and IVUS 45MHz single element transducer back-to-back at the same axial position, this probe can provide automatically co-registered, co-axial OCT-IVUS imaging. To demonstrate its capability, 3D OCT-IVUS imaging of a pig’s coronary artery real-time displaying in polar coordinates, as well as images of two major types of advanced plaques in human cadaver coronary segments, were obtained using this probe and our upgraded system. Histology validation is also presented.

 

Abstract Title: Biconically tapered fiber biosensor for label-free rapid immunoassays

Abstract: A biconically tapered optical fiber is a simple and cost-effective refractive index sensor based on modal Mach-Zehnder interferometry. For biosensing, the surface of the taper is typically functionalized with antibodies specific to an antigen to be detected. The antibody-antigen reactions create a biological nanolayer on the surface of the tapered region, which modifies the waveguide structure resulting in a phase difference between the propagation modes. As a result, spectral shifts are measured in the transmission spectrum of the sensor. Unlike biosensors that work based on fluorescent labels, the data can be recorded in real time enabling the study of the kinetics of antibody-antigen binding. In all previous studies, BTOFs based on Mach-Zehnder interferometry were reported in straight geometry, which cannot be used as a dip probe that can be immersed in solutions because of geometrical limitation. In addition, when there is a temperature increase, the substrate of the taper expands and the tensile stress on the sensor increases leading to breakage and increased temperature sensitivity. We report U-shaped biconically tapered optical fibers for label-free immunoassays. The tapered regions of the sensors were functionalized by immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations in the range 500 ng/mL to 50 microgram/mL. The limit of detection was estimated to be less than 500 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of antibody-antigen reaction is proposed as a rapid detection method.

Abstract Title: In Vivo Evaluation of Electrospun Polycaprolactone Graft for Anterior Cruciate Ligament Engineering

Abstract: The anterior cruciate ligament (ACL) is a band of anisotropic, dense connective tissue that connects the femur and tibia and is critical for the knee’s structural stability. ACL rupture is a common ligamentous injury that often necessitates surgical intervention and accounts for over $1 billion in annual costs in the United States. The current reconstructive methods use autologous or allogeneic tissue but suffer from drawbacks including donor site morbidity and limited supply, leaving opportunity for the exploration of a tissue-engineered substitute. We examine the use of a biomaterial, polycaprolactone, to serve as a platform for ligament reconstruction in a rat model. Electrospun PCL grafts were fabricated, laser cut, and coated with either collagen or heparin along with basic fibroblast growth factor, human foreskin fibroblasts, or both. Immunohistochemistry specific for macrophage infiltration indicates minimal inflammatory responses at 8 and 16 weeks postoperatively. Histological analysis of collagen reveals noticeable collagen deposition by week 8, and scaffold degradation followed by aligned scar tissue deposition by week 16. Mechanical testing data shows a trend of increasing stiffness and peak load to failure from day 0 to week 16, indicating that the polymer approaches native ACL properties. An overall trend of decreased inflammatory response, increased collagen deposition, and increased scaffold strength with time is observed, showing the potential for a ligament engineered approach to treat ACL rupture.

 

Abstract Title: The Third Dimension, Mechanics and Mammary Morphogenesis

Abstract: 1 out of 4 women diagnosed with breast cancer are over-treated, highlighting the deficiency in understanding of the disease for better detection and treatment. Recently, more care has been afforded to matrix mechanics given the positive correlation between breast tissue stiffness and cancer. However, to investigate the actual relevance of mechanical hypothesis in tumor development, it is important to choose a relevant in-vitro model that does not preclude matrix mechanics. We demonstrate that the current 3D culture standard of liquid overlay (bed of rBM underneath cells, with 2% rBM in media above) results in a mechanical environment which is not physiologically relevant and displays significantly different phenotype in comparison to the more relevant embedded method for breast cancer models. Whereas the former only consists of a 3D chemical but a 2D mechanical environment, the latter consists of a 3D chemical and mechanical environment. Our results display staggering differences in acinar behavior between the overlay and embedded methods and accentuates the importance of testing mechanics properly. The current overlay method does not recapitulate physiological conditions and provides a quasi-mechanical state. More importantly, these results suggest the need to reevaluate the underlying assumptions of the role of matrix mechanics in tumor progression in the framework of a relevant 3D model. Lastly, our results may extend out to other cell types whose behavior in 3D is studied using the overlay method, such as stem and other cancer cells.

Abstract Title: MicroTsunamis: a method for high-throughput screening of cellular mechanotransduction using laser microbeam generated cavitation bubbles

Abstract: No methods exist for the high-throughput screening of mechanotransduction. While sophisticated imaging cytometry technologies exist for rapid assessment of cellular activity, they cannot apply precise mechanical stimulation to cells at high-throughput. Moreoever, established methods to apply mechanical stimulation such as AFM, optical tweezers, or microfluidics are not easily integrated into conventional cytometry platforms and require specialized expertise. Thus, we aim to provide a novel method for high-throughput assessment of cellular mechanotransduction using laser generated cavitation bubbles (microTsunamis) with imaging cytometery employed in drug discovery.

 

Abstract Title: Billion-Voxel Optical Tomography using Lensfree Holographic On-Chip Imaging

Abstract: Optical microscopy is an irreplaceable technique for biomedical sciences. Conventional configurations of optical microscopes rely on lenses, which bring trade-offs such as magnification vs. field-of-view (FOV); throughput vs. compactness; performance vs. cost. Lensfree holographic microscopy overcomes such tradeoffs by adopting unit-fringe magnification configuration, which takes advantage of the rapid evolution of image sensor technologies, providing us cost-effective sensors with small pixel pitch and large pixel count. For imaging transparent objects, using a source-shifting based pixel super-resolution algorithm our holographic microscopy platform can reach a numerical aperture (NA) of >0.9 across a very large FOV of e.g., >20 mm^2.

Using this computational imaging modality we performed optical tomography by scanning the illumination angle across two axes, both spanning ±50 degrees. Pixel super-resolved holograms are synthesized to reconstruct the projection of the same sample at each illumination angle. Combining all these projections allows one to retrieve tomographic structures of the object. At 530 nm illumination, this lensfree tomographic modality achieves a lateral resolution of 0.35 µm and an axial resolution of ~ 2 µm, which corresponds to a voxel size of 0.03 µm^3. For a sample volume of ~5 mm^3, our imaging system provides voxels counts >150 Billion. This performance has been demonstrated by on-chip imaging of micro-particles as well as a wild-type C. elegans nematode (see the Figure)

Abstract Title: Enumeration of mRNA Transcripts from Single Cells Using a Microfluidic Device

Abstract: Accurate measurement of RNA transcripts from single cells will enable the precise classification of cell types and characterization of the heterogeneity in cell populations that play key roles in normal cellular physiology and diseases. As a step towards this end, we have developed a microfluidic device and methods for automatic hydrodynamic capture of single mammalian cells and subsequent immobilization and digital counting of polyadenylated mRNA molecules released from the individual cells. Using single molecule fluorescence imaging, we demonstrate that polyadenylated mRNA molecules from single cells can be captured within minutes by hybridization to polydeoxyribothymidine oligonucleotides covalently attached on the glass surface in the device. Our technology opens up the possibility of direct digital enumeration of RNA transcripts from single cells with single-molecule sensitivity using a single integrated miniaturized device.

Abstract Title: SHRINK-INDUCED SILICA STRUCTURES FOR ENHANCED FLUORESCENCE DETECTION FOR DNA MICROARRAYS

Abstract: We describe a manufacturable and scalable method for fabrication of multi-scale wrinkled SiO2 structures on shrink-wrap film for far-field fluorescence signal enhancements in DNA fluorescence microarrays. The development of DNA microarrays has proven invaluable for disease diagnosis. Conventionally, DNA microarrays are fluorescence based DNA microarrays because they offer numerous benefits, such as high sensitivity and multiplexing capabilities. Despite these advantages, a persistent challenge is to improve the detection sensitivity. Strategies to increase the fluorescence sensitivity of DNA microarrays include increasing the amount of capture probes or enhancing the fluorescence signal using noble metal nanostructures. However, oversaturation of probe density can also cause a reduction in the target DNA binding efficiency and metallic structures typically require precise and sophisticated equipment to yield near-field metal enhanced fluorescence (MEF) effects in heterogeneous areas of ‘hot spots’. Recently, Lin et. al reported generating silica, SiO2, structures from a pre-coated thermoplastic polyolefin (PO) shrink film (PO-SiO2) to enhance the fluorescence signal of bound biotin-streptavidin-Tetramethylrhodamine isothiocyanate (TRITC) biomolecules. In this abstract, we expand upon this strategy and demonstrate its applicability to DNA fluorescence microarrays. We performed a microarray by linking amine-modified single stranded (A30 ssDNA) onto an activated SiO2-coated PO film and hybridized with the complementary ssDNA to A30 tagged with Cy3 fluorophore. We then shrunk the entire substrate in less than ten seconds to generate SiO2 structures and produced a miniature microarray with enhanced fluorescence signal. Notably, our SiO2 structured substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM).

Abstract Title: Fluorescent Flatbed Scanner: An Ultra-Large Field-of-View Gigapixel Fluorescent Imaging System

Abstract: We report a cost-effective approach that is based on a flatbed scanner to image fluorescent microobjects over an ultra large field-of view (FOV) of 532 cm^2 [1]. We modified both the hardware and software components of the scanner, augmented it with an external LED array to provide the fluorescent excitation light in a dark-field configuration, and added a custom-designed emission filter to reject the excitation light scattered by the sample. We also reprogrammed the scanner’s driver to maximize its sensitivity, exposure time and gain to successfully detect and count fluorescent microobjects. The detection accuracy of this system is 98.8%, and the scanning of the total sample volume (~2.2 mL) is performed in less than 5 minutes. This fluorescent scanner is also capable of detecting fluorescently labelled cells, which was demonstrated by imaging white blood cells stained with Ethidium Bromide. This flatbed scanner based fluorescent imager has an optical resolution of 7.8 µm × 12.4 µm over a FOV of 280 mm × 190 mm, which yields an effective pixelcount of 2.2 Gigapixels. In conjunction with large area microfluidic chips, this ultra large field-of-view scanner system can perform high-throughput fluorescent imaging, which might be useful for cytometry applications and rare cell research.

[1] Z. Göröcs, et al. Lab Chip, (2013),13, pp. 4460-4466, DOI:10.1039/C3LC51005K.

 

Abstract Title: Rapid, single bacterial detection from blood using microencapsulated sensors

Abstract: The high mortality of blood stream infections is associated with the ineffectiveness and time-consuming process of bacterial detection and treatment. Unfortunately, blood culture, the gold standard for the detection of bacteremia, takes several days to obtain results. New molecular diagnosis methods, such as polymerase chain reaction (PCR), are often not sensitive enough to detect bacteria that occur at low concentrations in blood (1-100 colony-forming unit (CFU)/mL). Moreover, all these techniques are sophisticated and expensive, and therefore not well-suited for routine testing. Therefore, simple methods are urgently needed for rapid and sensitive identification of bacteria in blood, which has the potential to significantly reduce the mortality rate and the cost of medical care associated with blood stream infections.In this study, we have developed a system that detects bacteria in patient blood at single-cell sensitivity within a few hours. Our system integrates bacterium-detecting DNAzyme sensors, which are obtained by in vitro selection, with droplet microfluidics. Our central hypothesis was that the confinement of bacteria in droplets significantly increases the concentration of released target molecules that can be detected by the DNAzyme sensors in a rapid, real-time fashion. Specifically, infected patient blood was mixed with DNAzyme sensor solution, including bacteria lysis buffer, within the microfluidic channel, which was encapsulated in millions of individual picoliter droplets. Because bacteria exist at low numbers in blood, we anticipated each droplet will contain one or no bacteria. DNAzyme sensors fluoresced instantaneously in the droplets that contain bacterium. The droplets were monitored by APD (avalanche photodiode) embedded confocal microscopy in a high throughput manner. Our rapid detection and early intervention will therefore significantly improve the chances of treating blood stream infections and reduce mortality.

Abstract Title: MECs: Building blocks for microfluidic instruments

Abstract: The spread of microfluidic technologies into new application areas has been slowed by two main deficiencies. First, most existing microfluidic chips are single-purpose, and creating a new microfluidic tool for a new application typically requires that a whole new chip be designed and fabricated, a time-consuming process. And second, making microfluidic chips requires specialized training and access to fabrication equipment. As a result, many scientists and engineers may recognize the need for a microfluidic instrument in their research but are unable to build one. To address these deficiencies, we have created a set of “building blocks” which any scientist or engineer can literally “snap together” to create custom microfluidic instruments. We call these building blocks Multifluidic Evolutionary Components or MECs. “Multifluidic” indicates that MECs operate on multiple volume scales (from nanoliters to milliliters) and include functions from multiple fields (not only fluidics but also electronics, optics, and mechanics), and “evolutionary” conveys the ease with which new components can be designed and added to the library of existing MECs.Each MEC performs a fundamental function in a fluidic instrument, like controlling fluid flow, storing reagents, or mixing fluids. By arranging MECs symbolically in a schematic, then using the schematic to guide assembly of the MECs, a complete functional instrument can be built. For applications that require a custom microfluidic component, the socket MEC allows virtually any microfluidic chip (made using any process) to be packaged and plugged into other MECs. This enables custom microfluidic chips to easily leverage (and eventually be integrated into) the large library of MECs.By enabling researchers without specialized training or access to fabrication equipment to build custom fluidic instruments, MECs can enable applications for microfluidics throughout the biological and health sciences.

 Abstract Title: Optical Flow Paired With Machine Learning for Increased Detection of Drug-Induced Cardiotoxicty in Human Induced Pluripotent Stem Cell Derived Cardiomycoytes

Abstract: Present drug screening methods are incomplete in their ability to detect cardiotoxcity as 30% of drug attritions are attributed to drug-induced cardiotoxicity. Although the potential of human pluripotent stem cells-derived cardiomyocytes (hPSC-CM) as an in vitro model of the heart for drug screening has been shown, current methods with hPSC-CMs are not suitable for high-throughput. Methods that look at calcium flux are commercially available; but, they are susceptible to errors as certain drugs affect proteins involved in contraction mechanisms. We have previously demonstrated a platform that measures cardiotoxicity by analyzing bright-field (BF) images of hPSC-CMs. We aim to pair that platform with machine learning (ML) and compare its efficacy to a control method that examines calcium transients (CT). We hypothesize that ML can provide better accuracy as several parameters can be simultaneously evaluated and a binary response to drug-induced cardiotoxicity is provided. An hPSC line transfected with GCaMP-6, a calcium indicator, was used for both methods. To obtain CT signals, a GFP filter was used as GCaMP-6 fluoresces in the presence of calcium. The BF images were processed with an optical flow algorithm to generate vectors representing motion of the hPSC-CMs. Contractile profiles derived from the first principal component analysis of vectors were then classified with a ML algorithm. We examined the effect of E-4031, a drug that prolongs the QT interval. For the CT method, the lowest concentration at which cardiotoxicity was detected was 10 nM. With the BF method, 10 nM achieved an accuracy higher than 95% (96.3%), the designated cut-off for cardiotoxicity. Interestingly, 5 nM produced an accuracy of 94.8% with the BF method. Although it is below the cut-off, 5 nM may have an effect on the hiPSC-CMs. Thus, the BF method is at least comparable to the CT method and possibly more accurate. To further evaluate the efficacy, additional compounds are being screened.

 Abstract Title: A method for in vivo measurement of oxygen tension within subcutaneous devices

Abstract: Objective:Encapsulated cells often have low viability, resulting from low oxygenation in the implant site, together with reduced oxygen diffusion arising from distance from the capsule surface to the cells inside, creating hypoxic and anoxic core. Direct measurement of oxygen levels within encapsulated devices has not been reported, and it is yet unknown that hypoxia is the primary cause of cell death. Here, we are reporting a method of measuring oxygen within alginate capsules in vitro and in vivo non-invasively.

Method:Oxygen-sensitive microparticles were fabricated comprising an oxygen-sensitive dye embedded within a polystyrene matrix. 3% UPLVM alginate (Novamatrix) beads containing oxygen sensors were made using encapsulator machine (Nisco), cross-linked by 120 mM CaCl2 (J.T Baker). We measured oxygen content of the beads by feeding different percentage of oxygen gas to multi-well plates containing the encapsulated probe in vitro. Encapsulated probes were then implanted subcutaneously in rat (Sprague-Dawley), exposed to different percentages of oxygen for breathing gas, and then partial pressure of oxygen within the capsules were measured at various times post implant.

Results:In vitro results indicate that capsules are highly permeable to oxygen and are sensitive to oxygen levels throughout the clinical range. In vivo readings performed at various time-points post implantation show that the oxygen content within the implanted capsules changes as the implant site heals, and the dynamic delay between blood oxygen content and bead oxygen content also changes over time

Significance of impact:We have developed a technique for measuring the oxygen concentration continuously inside an implanted capsules .This measurement could have implications for identifying designs that lead to either cell survival or cell death. Also, we may be able to correlate that level with critical points in the wound healing process.

 

Abstract Title: Developing an Optical Biosensor in Plant Leaves

Abstract: Maize, one of the most widely grown staple crops of sub-Saharan Africa, suffers from significant loss due to biotic stresses from fungi, bacteria, and other pests and pathogens. Means to improve protection of maize crops, including the use of fertilizers and pesticides, are not widely employed by smallholder farmers in sub-Saharan Africa due to their high cost. Here, we report the initial development of a biodetection platform for maize crops to provide smallholder farmers with an effective tool for monitoring biological threats in the farm. By taking advantage of both the porous leaf surface structure and internal vasculature of plants, we adopt a biocompatible, needle-less syringe infiltration strategy to introduce functional biomolecules directly into leaves for lateral-flow detection of a mock pathogen marker, fluorescein. The design of our detection system incorporates the use of anti-fluorescein antibody-coated microspheres of optimal size to become easily infiltrated and subsequently retained in the leaf capillary and surrounding tissue. This enables the detection of specific target biomolecules (e.g., pathogens) within the plant in a non-invasive manner. Our detection platform is both effective and low-cost, exhibiting a limit of detection at 8µM fluorescein and costing approximately $0.75 for reagents per test. We are confident that this proposed technology will make a significant impact on improving the yield of healthy food crops by smallholder farmers across sub-Saharan Africa

Abstract Title: In vivo imaging of the human upper airway using long range optical coherence tomography

Abstract: Long-range optical coherence tomography (OCT) has the potential to provide high-speed three-dimensional tomographic images of the upper airway with high resolution and without the use of ionizing radiation. We present work on the advancements of our long range OCT system. Imaging is achieved thru the use of 3 types of endoscopic probes, 2 proximal rotation designs with outer diameters of 1.4 mm and 0.7 mm and 1 distal rotation design featuring a new short length micromotor. Imaging from the bottom of the larynx to the end of the nasal cavity is completed within 40 s.

 Abstract Title: Patterned Adhesive Construction of Nonplanar Three-Dimensional Paper Microfluidic Circuits

Abstract: Paper microfluidics is a rapidly expanding field developing low-cost diagnostic devices. In the most common devices, lateral flow, complex detection can require large footprints, or potentially incompatible additives to adjust fluidic timing. 3D devices can perform multiple simultaneous detections, housing complex channel geometries in a small footprint, but require external clamps, heavy adhesive application, or precisely aligned adhesive tape to ensure interlayer contact. Nonplanar 3D devices offer comparable capabilities to planar devices without requiring external clamps, and can potentially incorporate other capabilities, such as actuation.

This work details the application of adhesive through a perforated metal sheet to construct nonplanar 3D circuits from a single, folded sheet of paper. Such patterning reduces adhesive interference with fluid wicking along channels and between layers, while still providing enough adhesion to allow interlayer wicking. Circuits constructed with patterned adhesives are capable of being repeatedly unfolded (within the adhesive’s tack range) while maintaining interlayer contact upon refolding, which allows more complex folding. Circuits did not suffer any noticeable loss in wicking speeds or success rates up to 24 hours after assembly. Once used, circuits can be unfolded to view internally displayed results, allowing smaller sample volumes to be used, and provides a method of keeping sensitive results private.

In this study, a combination of an adhesive pattern and channel size was identified that resulted in planar circuits able to wick fluid throughout their length and between layers. This knowledge was then used to construct a nonplanar 3D circuit (within an origami peacock) capable of wicking three colored solutions across multiple layers without mixing. Such nonplanar 3D structures are expected to offer a new platform for exploring new circuit functions and designs.

 

Abstract Title: Ultra-high throughput isolation of circulating tumor cells with microfluidic Vortex technology

Abstract: Here we describe advances in microfluidic Vortex trapping technology for the rapid, size-based isolation of circulating tumor cells (CTCs) from blood. CTCs are extremely rare but show promise as minimally invasive biomarkers for cancer patient prognosis, and treatment monitoring. Whereas current isolation approaches have primarily focused on achieving high efficiency of capture, techniques are limited by throughput and purity.The High Throughput Vortex Chip (Vortex HT) is a parallelized PDMS microfluidic chip with 192 rectangular trapping compartments. At a high flow rate (800 µL/min of whole blood), large cells (>15 µm diameter) experience large inertial shear gradient lift forces and become trapped in laminar fluid microvortices that develop in the reservoirs. Smaller blood cells do not experience a sufficient lift force and are washed away. By lowering the flow rate, vortices dissipate to release viable cells in a concentrated volume (~300 µL) for downstream analysis.

Vortex HT separates MCF7 breast cancer cells from blood with 10% higher efficiency than the previously described Vortex Chip, while maintaining high sample purity (>80%). At double the flow rate (8 mL/min of 10X diluted blood), the waste from the first trapping run can be re-processed through the device to achieve higher capture efficiency (~2X) in the same total run time, while subsequent re-processing further increases efficiency with a trade-off of increased processing time. Cells are released in a low volume (300 µL) and maintain ~80% viability with the capability of being cultured off-chip. Finally, the device demonstrates high CTC capture from clinical blood samples from non-small cell lung cancer patients.

The high throughput isolation of viable CTCs with Vortex HT is a rapid, convenient sample preparation technique that allows for flexible downstream investigations, such as molecular analysis, immunohistochemistry, cell culture, or other diagnostic and biological studies.

 Abstract Title: ARRAYED FORCE PHENOTYPING OF SINGLE-CELLS FOR HIGH-THROUGHPUT SCREENING AND ANALYSIS

Abstract: The ability of cells to apply traction forces, herein termed contractility, varies in magnitude across different cell types, changes with cellular differentiation and is necessary for preservation of health in organisms. As such, contractility may serve as a label-free biomarker that can be used to classify cell phenotype and probe cell state. We describe a high-throughput platform to assay contractility of single-cells that is composed of arrays of uniformly shaped adhesive molecular patterns that deform in response to cell-induced forces.Currently, cell contractility is studied primarily using either traction force microscopy (TFM) of substrates with embedded fluorescent particles, or elastomeric micro-post arrays (EMA), however, both approaches are limited in through-put and lack standardization in data from cell to cell.Unlike these methodologies, our platform uniformly confines cells to defined shapes designed to focus their traction forces to specific points creating well-defined deformations in the ultra-soft substrates that can be characterized with a single measurement, allowing for direct comparisons between cells.The platform consists of an ultra-soft layer of PDMS patterned with fluorescent adhesive molecules such as extracellular matrix (ECM) proteins that is supported by a glass substrate. Seeded cells adhere to the adhesive patterns, exert traction forces, and deform the patterns. Fluorescent microscopy is used to image the resulting patterns and custom-written image analysis software measures the dimensions of the deformed patterns.

Abstract Title: Enhancing the Phase Separation Behavior of a Micellar Aqueous Two-Phase System in a Paper-Based Diagnostic

Abstract: The lateral-flow immunoassay (LFA) is an attractive method for point-of-need disease diagnosis due to its ease of use, rapid processing, and minimal power and equipment requirements. Our lab previously improved the sensitivity of LFA by using a Triton X-114 micellar aqueous two-phase system (ATPS) to concentrate a target protein prior to detection via LFA. The combination of ATPS and LFA successfully lowered the LFA detection limit for a model protein transferrin by 10-fold. However, the micellar ATPS phase separates on the order of hours in a test tube, rendering it impractical for point-of-need applications. Moreover, the previous system required trained personnel to extract the phase containing the concentrated biomarker. An effective device would require a reduction in the ATPS phase separation time and the elimination of an additional extraction step without sacrificing the improvement in LFA sensitivity. In this study, we applied the ATPS sample directly into a paper-based membrane and discovered that we can significantly reduce the phase separation time to a matter of minutes, while also removing the need for an extraction step. The paper set-up takes advantage of fiberglass material arranged in a 3-D architecture to concentrate a target molecule as it flows. This is the first time that a paper-based device has been used to enhance the phase separation of a micellar ATPS, and we demonstrated its applicability by coupling it with LFA to detect transferrin. The integrated paper-based micellar ATPS and LFA device was able to detect transferrin at levels that were 10-fold lower than that of LFA alone (Figure 1). In summary, the integrated device improves LFA sensitivity in a relatively short time without the need to extract phases, and therefore has the potential to become the next generation diagnostic.

Abstract Title: Micro-tools to study single mitochondrial membrane potential and respiration.

Abstract: Mitochondria play a multitude of roles in cell fate. If in cell life, they provide the majority of ATP needed for cellular processes, in cell death, the organelles are a decider of apoptosis or controlled cell death. As a result, damages to the mitochondrial functions have detrimental effects and research has substantially linked mitochondrial dysfunctions to diseases such as Leber’s hereditary optical neuropathy, Parkinson’s diseases, and cancer. Studies on the relationship between mitochondrial dysfunctions and diseases typically report two key parameters: the mitochondrial inner membrane potential and respiration, which together reflect the functional status of the mitochondrion, namely its capacity to do work and its metabolic state and intactness. However, current assays require significant amounts of sample size and measure only the average response from an ensemble of mitochondria, thereby obscuring single mitochondrial behaviors. Our research aims to study single mitochondrial membrane potential and respiration with micro-fabrication. We developed a fluidic platform with nanoscale dimensions that can trap single mitochondria for membrane potential assay by fluorescence microscopy. This platform overcome issues often encountered in studying mitochondrial membrane potential such as background fluorescence, photobleaching, mitochondria movement out of the plane of focus, and difficulties in precise delivery of substrates. For mitochondrial respiration, we are working on microchambers that can sense the oxygen consumption rate from a single mitochondrion or a single cell. In addition to lowering the sample size for sensing oxygen consumption, we are also aiming for cost-effective and high-throughput technology.

Abstract Title: Amperometric Immunosensor for Detecting Growth Hormone Use by Athletes

Abstract: Human growth hormone (GH) is produced by the anterior pituitary gland and promotes growth of tissue through direct uptake at target tissue sites, or alternatively, by regulating production of insulin-like growth factor-1. The World Anti-Doping Agency considers GH a performance enhancing substance so the use of GH by athletes is prohibited in most sports. The current immunoassay for GH detection is suboptimal for routine screening of blood samples because of the large resources required for collecting, processing, transporting (blood must be shipped to testing sites at controlled temperatures), and testing of blood samples.

The proposed research utilizes an amperometric sensor system with polypyrrole-streptavidin surface, and a biotinylated capture antibody and peroxidase-conjugated reporter antibody to measure GH isoforms in whole blood samples. This method reduces the need for sample processing, requires less sample volume, and reduces assay time. The method selectively targets the 22kD and 20kD isoforms of GH, which will provide more statistical discriminatory power than the existing GH test. Additional explorations are being made into the application and optimization of low-voltage, low-frequency applied voltages for enhancing sensor speed and performance.

The present results demonstrate that the amperometric immunosensor assay configuration can detect GH isoforms in 10µL of whole blood with acceptable linearity (R^2 = 0.94) and a limit of quantitation of 20 pg/mL. The method also reduces assay time (compared to the current GH isoform ratio test) by approximately 40%. Further testing will be performed to determine if the method can be used tor measure GH isoforms in dried blood spots. The viability of a low-voltage electric field based method will also be evaluated for additional sensor speed.

 Abstract Title: Portable Integrated Frequency Domain and Continuous Wave Real-time Diffuse Optical Spectroscopy and Imaging

Abstract: In this work, we present a portable (10cm×5.5cm×3.25cm) high speed integrated frequency-domain and continuous wave (CW) system for spectroscopic imaging in diffuse media. This system measures four tissue chromophore concentrations (water, lipid,deoxygenated and oxygenated hemoglobin) at eight near-infrared wavelengths ranging from 660nm to 980nm, in real-time. The frequency domain (FD) module measures the phase and amplitude of diffusing light from 50-250 MHz with an operating speed of 2Hz while the CW module operates at 80Hz. The FD component provides quantitative information by separating scattering from absorption using the acquired phase and amplitude data. The CW component expands spectral bandwidth and improves acquisition speed by measuring only amplitude changes. The CW system has 110 dB dynamic range, enabling measurements in tissue with a source-detector spacing up to 4.5cm. The CW module frequency-encodes wavelengths for parallel illumination resulting in rapid data acquisition. It is also less sensitive to background noise from ambient light by utilizing low-frequency modulation and a narrow bandpass filter on the source and detector sides, respectively. The standalone FD module can be utilized in applications where scattering coefficients are required from every measurement and only deoxygenated and oxygenated hemoglobin concentrations are desired. In combined mode, concurrent FD and CW measurements are available where information about water and lipid contents is also necessary. In media with constant scattering, a single FD measurement can be used as a baseline and the CW module can be used for subsequent measurements to extract the absolute chromophore absorption coefficients. Finally, if only relative changes in tissue content,(e.g., oxygen saturation) are desired, the instrument can operate in standalone CW mode. We will demonstrate the performance of this combined system using a tissue-simulating (Breast tissue) phantom with an embedded inclusion.

Abstract Title: Combined Optical Tweezers and Broadband Quantitative Phase Microscopy for High Resolution Characterization of Lipid Bilayer Nanostructures

Abstract: Various cellular processes such as motility, division, transport, and endocytosis involve a change in membrane shape. These shape changes accentuate the role of membrane and cytoskeleton mechanics, and the dynamic interactions between these two cell components. Tether formation from cell membranes provides a technique to quantify membrane mechanical properties and membrane-cytoskeleton interactions. We have combined optical tweezers with spatial light interference microscopy (SLIM) to simultaneously form lipid bilayer nano-tubes (tethers) from cells and measure the nano-scale diameter of the tether. Accurate measurement of the tether diameter is relevant to quantification of membrane tension, bending modulus, and adhesion energy of the membrane-cytoskeleton structure. The SLIM module produced a phase map of the sample where the phase values are correlated to sample thickness along the illumination axis (z), and the refractive index (RI) mismatch between the sample and the surrounding media. We used beads with 4 µm diameter to pull lipid bilayer tethers from endothelial cells (cell culture medium RI=1.337) using the optical tweezers apparatus, and imaged the tethers using the SLIM module. The RI of the tether, as estimated by correlating the lateral size measurements from bright field images and the corresponding phase measurements, ranged from 1.354 to 1.368. Our SLIM imaging system provided a resolution in tether thickness that ranged between 21 nm and 38 nm. Our integrated platform also provides the ability to simultaneously manipulate and image cell organelles in a non-contact and marker-free manner at nanometer spatial resolution.

Abstract Title: Wafer-scale Titanium Anodic Bonding for Microfluidic Applications

Abstract: Recent advances now allow deep reactive ion etching of titanium at the micro- and nanoscale (i.e. Ti DRIE), which therefore creates new opportunities for realization of robust, high-pressure microfluidic devices. However, the intrinsic opacity of Ti precludes optical interrogation within such devices, thus motivating the development of means for heterogeneous bonding with transparent substrates. Anodic bonding, which is used widely for more conventional silicon-based microfluidics, represents a promising option in this regard, due to the rigidity, chemical and thermal stability, and well-known surface properties of typical glasses, coupled with the potential for achieving high-strength, hermetic bonding at relatively low temperatures. However, to date, there has been only limited study of Ti/glass anodic bonding for microfluidic applications, particularly for bulk substrates at the wafer-scale.Herein, we report the development of an anodic bonding process that allows, for the first time, high-strength joining of bulk titanium and N-BK7 glass substrates at the wafer-scale, without need for interlayers or adhesives. Uniform, full-wafer bonding is achieved at temperatures as low as 250 °C, and burst pressure testing reveals that failure occurs via crack propagation through the glass, rather than the titanium/glass interface, thus demonstrating the robustness of the bonding. Using optimized bonding conditions, we demonstrate the fabrication of a rudimentary titanium/glass-based microfluidic device and its leak-free operation under pressure-driven flow.We also demonstrate the potential for monolithic integration of nanoporous titanium dioxide within such devices, thus illustrating the promise embodied in titanium anodic bonding for future realization of robust microfluidic devices for photocatalytic applications.

Abstract Title: Bimodal imaging of atherosclerotic plaques using fluorescence lifetime imaging (FLIm) and intra-vascular ultrasound (IVUS)

Abstract: Sudden cardiovascular events such as heart attack or stroke are caused due to rupture of atherosclerotic plaques in arteries. While angiography is the standard imaging technique during percutaneous coronary intervention (PCI), it provides limited information about the nature of the plaque occluding the vessel. The goal of this study is to combine two imaging techniques, intra-vascular ultrasound (IVUS) and fluorescence lifetime imaging (FLIm) into a hybrid catheter to simultaneously probe structure as well as biochemistry of arterial vessels for improved plaque assessment. 10 ex-vivo human coronaries were imaged using a prototype catheter that includes a rotational 300 µm side-viewing fiber-optic (excitation source – Fianium, 355 nm, 10 KHz, 1.8 mJ/cm2) integrated with a conventional IVUS (40 MHz, Boston Scientific). Autofluorescence was measured at three emission wavelengths (CH1- 390/40, CH2- 452/45, CH3- 542/50 nm) corresponding to emission from collagen, elastin, and lipoproteins respectively. Pathological features including fibroatheroma (FA), fibrocalcification (FC), and inflammatory cell infiltration (CD45+, CD68+) were identified in histology and the corresponding co-registered FLIm and IVUS data were studied. FLIm results showed lower lifetime values for lipid rich FA (CH1, 3.47±0.23 ns, CH2, 3.67±0.20 ns) compared to collagen rich fibrous tissue (CH1, 4.17±0.27 ns, CH2, 4.41±0.79 ns). Additionally, lifetime values also showed a difference between inflamed FA (CH1, 3.40±0.23 ns, CH2, 3.53±0.16 ns) and FA without inflammation (CH1, 4.13±0.30 ns, CH2, 4.01±0.56 ns). Integrated backscatter values from IVUS radio-frequency data were able to distinguish between hyperechoic fibrocalcified (-61.4±6.8 dB) and hypoechoic lipid-rich (-81.1±4.5 dB) regions. Results demonstrate the ability of FLIm to complement IVUS imaging in characterizing plaque composition for intravascular diagnosis that may assist a physician in guiding PCI.

Abstract Title: Exploring the spectral properties of genipin reagent and its reactions with collagen

Abstract: Genipin is an active compound found in the Gardenia fruit extract that is an excellent cross-linking reagent for proteins. Also, it has low acute toxicity and is environmentally friendly.

A cross-linking reaction of genipin compound with amines in amino acids of proteins generates a blue pigment, where exact color is determined by the reacting amino acid. When we treat the translucent collagen hydrogels with clear solutions of genipin, a noticeable color develops within 24 hours at room and higher temperatures. Additionally, gels become highly fluorescent as a result of this cross-linking reaction.We imaged second harmonic generation (SHG) and two photon fluorescence (TPF) signals from collagen fibers within genipin cross-linked hydrogels at a single excitation wavelength of 800 nm. The two-photon spectra illustrates that in genipin-modified materials, 800 nm excitation wavelength generates TPF centered within the green (approximately 490 nm center) as well as red (approximately 615 nm center) spectral regions. The ratio of the intensity of 615 nm emission band to the intensity of 490 nm band is 5.3 at 24 hr post modification at 37 C and does not appear to change significantly upon longer (72 hrs) modification times. There is a very slight bathochromic (red) shift when the excitation wavelength varies between 760 and 900 nm.Quantification of SHG signals from genipin-modified and control collagen hydrogels showed that after 24 hours of modification at 37 C the former generated approximately 4 times less SHG compared to the latter. SHG contrast helped to identify spatial alterations to the microstructure of collagen hydrogels as a result of genipin cross-linking reaction. The 5-10 µm long “fiberlike” structures within the collagen hydrogels became ‘washed out’ upon cross-linking likely due to lowered intensity of SHG signal associated with them. Presently we also explore the formation and cross-linking reactions of polymeric genipin structures.

 Abstract Title: Manipulating gold nanoparticles to achieve effective and rapid detection of protein biomarkers for resource-poor settings

Abstract: The lateral-flow immunoassay (LFA) is a paper-based detection method that can become ideal for resource-poor settings since it is rapid, inexpensive, easy to use, and portable. However, its sensitivity is lower than that of traditional lab-based assays. In this study, an aqueous two-phase system (ATPS) comprised of polyethylene glycol (PEG) and potassium phosphate salt was investigated to concentrate the model protein transferrin (Tf) prior to its detection. In order to concentrate Tf effectively in an ATPS, the target protein must partition, or distribute, extremely into a small volume. However, due to its relatively small size, the protein partitions fairly evenly between the two phases of an ATPS. To address this problem, we developed two types of antibody-decorated gold nanoprobes that achieved the following functions: (1) maintained stability in the high salt environment of our ATPS; (2) captured the target protein in the sample; (3) partitioned extremely to either the bottom PEG-poor phase or the interface; (4) served directly as the colorimetric indicators for LFA after being extracted and applied to the paper strip. To capture and drive Tf to the bottom PEG-poor phase, dextran-coated gold nanoprobes (DGNPs) were used. Dextran did not interact favorably with the greater number of PEG molecules in the top phase due to being more hydrophilic, and the relatively large DGNPs also experienced greater steric, repulsive excluded-volume interactions with the more abundant PEG molecules in the top PEG-rich phase. This resulted in the extreme partitioning of DGNPs into the bottom PEG-poor phase. Alternatively, we also employed PEGylated gold nanoprobes (PGNPs) to capture and drive Tf to the interface between the two bulk phases. PGNPs partitioned extremely to the interface due to a balance between the attractive PEG-PEG interactions and the repulsive excluded-volume interactions with the greater number of PEG molecules in the top phase.

Abstract Title: Developing an algorithm to enable rapid characterization of alginate microcapsules

Abstract: Islet transplantation, while having been demonstrated to be a temporary cure for Type I Diabetes, has been problematic due to transplant rejection by the recipient’s immune system thus requiring chronic immunosuppression. Islet microencapsulation, where the islets are coated with a biocompatible polymer using air-driven or electrostatic microcapsule generators, has demonstrated various degrees of success in small and large animal trials. This success however, depends greatly on microcapsule morphology, the degree of islet coverage and the placement of the islets within the encapsulating hydrogel. Since hundreds of thousands of microcapsules are generated during the process, characterization of encapsulated islets without the help of some degree of automation would be painstakingly difficult, time consuming and error-prone due to inherent observer bias. In order to address these issues, we have developed an algorithm that can analyze hundreds of microencapsulated islet and characterize their size, shape, circularity, and distortion with the help of ImageJ. Using the conventional method, three independent observers required 11 min 21 seconds on average to measure the diameter of 50 microcapsules, producing results with significant variants: 393±8 , 390±13 , 392±10 . When using the algorithm, the same results were obtained within 37±3 seconds, and produce results with zero variation: 406±11 . With this innovation, microencapsulated islets can be characterized with great consistency for pre-transplant assessment and quality analysis within a fraction of the time. This innovation will find wide applicability in the fields of islet and stem cell encapsulation and other microencapsulation processes by eliminating human error and reducing analysis time.

Abstract Title: An Electronic Bandage for the Early Detection of Pressure Ulcers in Vivo

Abstract: Each year, over 2.5 million Americans will develop pressure ulcers, costing more than $11 billion to treat annually. Pressure ulcers, which primarily affect bedridden and wheelchair-bound patients, are large chronic wounds caused by the restriction of blood flow to tissues near bony prominences of the body, such as the hip, ankle, and shoulder. Pressure ulcers are easily preventable simply by turning the patients and restoring blood flow before the affected tissue dies. However, current monitoring methods, such as photograhing and caretaker observation, are highly subjective and labor intensive. A fast, objective method of determining tissue health is critical to ulcer prevention. In response to this challenge, we are developing a novel, flexible monitoring device capable of real-time, quantifiable monitoring of high-risk areas. We have designed and implemented an electronic sensing device capable of mapping the complex impedance across a wound surface using an electrode array. The electronic bandage is fabricated using inkjet printing of gold nanoparticle ink onto a flexible PEN substrate. Impedance spectroscopy is used to measure and characterize tissue health, thus allowing physicians to objectively detect tissue deterioration and intervene before tissue necrosis. Preliminary data from a pilot study on a rat model demonstrate that our device can detect mild pressure-induced damage, even when the damage is not visually apparent. These results pave the way for the development of a true “smart bandage”, and have the potential to significantly improve patient outcomes by enabling earlier intervention for pressure ulcers and other chronic skin wounds.

Abstract Title: Multispectral time-resolved fluorescence spectroscopy system for real-time tissue diagnosis

Abstract: We report a rapid multispectral time-resolved fluorescence spectroscopy (ms-TRFS) technique that enables real-time tissue diagnosis through a single fiber-optic probe that can be handheld or integrated in robotic instruments. The ms-TRFS system allows for fast acquisition of fluorescence decays in multiple wavelength bands simultaneously. To demonstrate this concept, a fully automated ms-TRFS system based on a pulse sampling method was constructed. The system consist of a pulsed laser (700 ps pulse width, 30 Hz repetition rate), a custom wavelength selection module (4 spectral bands), a fiber-optic probe (600 µm core diameter), and a detection system using a fast response photomultiplier and digitizer (3 GHz bandwidth, 12.5 GS/s sampling rate). This system can provide up to 7.5 Hz continuous sampling rate of the ms-TRFS data. To achieve real-time display of diagnostic information, such system requires (1) a robust means of data acquisition to maintain an adequate signal-to-noise ratio during dynamic changes of fluorescence excitation-collection geometry and (2) and fast processing/display of fluorescence lifetime data. For this, we developed a close-loop control of fluorescence signal, based on modulation of fluorescence photon detection multiplication. Real-time ms-TRFS data analysis was achieved by implementing data processing algorithm in the system software platform. Proof-of-concept experiments were conducted in tissue phantoms and specimens to demonstrate potential applications of this technique. This includes guidance of stereotactic brain biopsy using fiber-optic needle probe, raster scanning of surgical margins using handheld probe, and tissue surface scanning with a fiber-optic probe integrated in a robotic surgical instrument. Current results showed the ability of this technique to display accurate fluorescence lifetime values and to discriminate different fluorescence markers and tissue types (e.g. epithelium, muscle and adipose tissue) in real-time.

 

Abstract Title: Spectroscopic Profiling and Metabolic Mapping of Cancer Cells using endogenous fluorescence

Abstract: Advances in hyper- or multi-spectral based imaging cameras have expanded new areas of cellular research for the last 20 years. Most recently, intensity signals can now be detected with a line spectrograph in which light is transmits over an imaging signal to an ultra sensitive CCD camera with fast readout speed. However, spectrally resolving these images is highly problematic given that most fluorescent emission spectra are broad. In our work, we used  Andor’s iXon Ultra EMCCD camera  to obtain spectral emission of cancer cell autofluorescence excited with a multiphoton laser source. In parallel we also performed fluorescence lifetime imaging microscopy (FLIM) to map free to bound NADH ratio distribution within single cells. In our approach the raw spectral and FLIM data sets are  transformed into sine and cosine components and plotted in a polar coordinate plot called the phasor plot. We can spectrally resolve intrinsic fluorescent species with nanometer resolution and map metabolic states of cells with the lifetime maps.The FLIM phasor distribution shows a clear difference between metabolic states of the nucleus and cytoplasmic regions. The nuclei had a larger fraction of free NADH indicated by a distribution shifted more forward the shorter lifetime as compared to the phasor distribution of the cytoplasm.  Using the spectral camera phasor analysis and FLIM we have been able to identify autofluorescence of cell membrane, mitochondria, nucleus and other organelles. Given the complexity of these chemical species, we treated the cells with various biochemical agents used to perturb metabolic states and cellular function to identify changes in spectroscopic signals.  A map of these chemical species can provide important information to identify proliferation, stress and dysplasia at the single cell level and can be applied to in the study of cancer and other diseases.

 

Abstract Title: Cortical Bone Laminar Analysis: A Novel Technique for the Quantization of Cortical Porosity in Clinical Imaging with HR-pQCT

Abstract: Cortical bone microstructure is vital to the mechanical integrity and fracture resistance of long bones. In particular, cortical porosity is responsible for over 75% of age-related reduction in cortical strength. However, studies have revealed that the current gold standard, Dual X-ray Absorptiometry (DXA), is insufficient as a diagnostic tool for the prediction of fractures. High Resolution-peripheral Quantitative Computed Tomography (HR-pQCT), with an isotropic voxel size of 82 µm, is capable of imaging the cortical bone microstructure in vivo. Thus, the aims of this study were to: 1) develop a novel technique to localize and characterize cortical porosity within laminae for HR-pQCT images; and 2) apply this technique to explore gender- and age-specific pore distribution. To address these aims, we performed a cross-sectional evaluation of HR-pQCT images of the distal tibia from 145 individuals (92 females, age = 47.8 ± 15.7 years; 53 males, age = 45.5 ± 16.3 years). Cortical bone laminar analysis localized pores to one of three cortical laminar layers: endosteal, midcortical, or periosteal. Porosity, pore size, and pore number were quantified within each layer across genders and age groups. Results indicate that gender-specific mechanisms of age-related cortical bone loss may exist. Overall detectable porosity was lower in young females than males, but female porosity increased more dramatically with age. Porosity increase in females was most pronounced in the midcortical layer, while porosity increase in men was more uniform throughout the cortex. In females, porosity increase was driven primarily by increased number of pores. In males, both number and size contributed to increased porosity with age. Measures of cortical porosity through HR-pQCT images may prove crucial in determining the underlying factors behind bone fragility as well as pinpointing possible targets for therapeutic intervention against metabolic bone diseases.

Abstract Title: Application of Quantitative FRET Technology for Km Determination of SUMO and ATP

Abstract: SUMOylation has important roles in many key physiological and pathological processes. The SUMOylation cascade involves a heterodimer of activating enzyme, E1 (Aos1/Uba2); a conjugating enzyme E2 (Ubc9); and many ligase enzymes, E3. Focusing on the activation step of the SUMOylation process, we examined the interaction of E1 with its substrates. Previous studies reported the Km of E1 enzymes in ubiquitin and other ubiquitin-like pathways. However, the Km of the SUMO paralogs (SUMO2 and SUMO3) is unknown. Here we used quantitative FRET to measure the SUMO E1 enzyme kinetics of SUMO-1, -2, and -3, and ATP under steady state conditions. We found that the enzyme kinetics from the quantitative FRET method are comparable to those for conventional radioactive assays. Additionally, we found that the kinetic constants, Km, of SUMO2 (3.418 ± 0.9131 µM) and SUMO3 (2.764 ± 0.75 µM) are about four to five times higher than SUMO1 Km (0.7458 ± 0.1105 µM). FRET technology has some disadvantages for determining Km, including the ability to monitor reaction progress in real-time with high-throughput and high-sensitivity and in an environmentally friendly assay. These results extend the utility of quantitative FRET in characterizing protein-protein interaction and enzyme kinetics.

Abstract Title: Computer Aided Design of ASiP as a Drug for Metastatic Melanoma

Abstract: Currently, a diagnosis of metastatic melanoma has a fatal prognosis with a life expectancy of 6 – 12 months. Melanoma has a unique feature in that it is resistant to chemotherapy because melanosomes sequester the treatment agents. For Melanocortin Receptor 1 (MC1R), a cell treated with alpha-Melanocyte Stimulating Hormone (a-MSH) will have greater melanosome production whereas treatment with Agouti Signaling Protein (ASiP) decreases melanosome production below basal levels. Here I combine the results of peer reviewed in-vitro biochemical experiments with computational modeling, sequence optimization, and binding prediction tools to engineer ASiP for optimal selectivity and binding affinity to MC1R. ASiP would function as a drug, in tandem with cisplatin, by reducing the number of melanosomes present in the cell, which would allow the chemotherapeutic to reach its intended target and trigger cell death. I am currently synthesizing the engineered ASiP mutant and will measure the mutants’ performance in-vitro using a proven competition assay for ASiP and MC1R. In-vitro performance will be compared against in-silico performance predictions to refine our methodology. If the engineered mutant shows superior in-vitro performance it will be synthesized in larger quantities and provided to our medical collaborators to measure the increase in cisplatin efficacy when delivered in tandem to treat metastatic melanoma.

 

Abstract Title: Smartphone-based Microscopy for Imaging of Single Fluorescent Nanoparticles and Viruses

Abstract: A field-portable fluorescence microscopy platform installed on a smartphone for imaging of individual nanoparticles as well as viruses is demonstrated. This lightweight imaging device consists of a compact opto-mechanical attachment (~186 grams) added to the existing camera module of the smartphone [1]. The attachment contains a light source for excitation, an emission filter, a collection lens, a sample tray, and a focusing stage. The samples are inserted from the side of the device and illuminated by the excitation beam at a high incidence angle (75o) to achieve a strong signal-to-noise ratio (SNR) in dark-field imaging. Using this cellphone based microscopy platform, single 100-nm fluorescent particles as well as individual fluorescently labeled human cytomegaloviruses (HCMVs) were detected, and their sizes were validated using scanning electron microscopy. This smartphone enabled single-nanoparticle fluorescence microscopy platform could be very useful for sensitive and specific detection of various biological targets such as labeled bacteria, virus particles, and potentially single molecules, and thus offers new opportunities for point of care (POC) diagnostics, for e.g., viral load measurements in remote or resource-limited environments.

Abstract Title: Self-assembled liquid nanolenses for wide-field nanoparticle and virus imaging

Abstract: The imaging of nano-scale objects, such as viruses, typically requires high-resolution electron or optical microscopes, which are expensive, bulky, and limited in field of view (FOV) to areas of order 0.01-0.1 mm^2. Small FOVs make it difficult to analyze low particle concentrations and limit the potential for rapid readout of multiplexed biomedical assays. Conversely, large-FOV imaging systems are in general not sensitive enough to detect the weak scattering signals from individual nanoparticles. Here we present two different label-free approaches that enable the detection of sub-100 nm particles and viruses across FOVs in the range 20 mm^2 to >10 cm^2, based on the fabrication of self-assembled liquid polymer nanolenses using either gravity-driven microfluidic flow or solvent evaporation. Both methods produce nanolenses with shapes quantitatively described by the Young-Laplace equation. These nanolenses reduce the detection threshold on particle size from approximately 250nm to below 100nm in both conventional low-magnification microscopes and field-portable lensfree holographic microscopes. Using these lenses, we have detected and enumerated individual H1N1 influenza viruses, adenoviruses, CpGV granuloviruses, and various other nanoparticles (see figure). Recorded optical signals correlate with particle size, providing a means to discriminate between different types of particles in a heterogeneous sample. These results form the foundation for new biomedical opportunities, such as the early detection and screening of viral diseases in resource-limited and field settings.

Abstract Title: SMARTPHONE ENABLED WATERBORNE PATHOGEN DETECTION USING FLUORESCENCE MICROSCOPY

Abstract: Rapid and sensitive detection of waterborne pathogens using a field-portable device is important for resource-limited settings and field conditions. Here we present the detection of Giardia lamblia cysts, one of the most common waterborne pathogens, in water samples using a cost-effective and handheld imaging system running on a smartphone. This pathogen detection platform is composed of 2 parts: a cellphone-based fluorescent microscope and a sample processing cassette. A custom-designed 3-D printed cellphone attachment, which includes excitation and emission filters, an external lens, light-emitting diodes and batteries, make up our fluorescent microscope design in connection with the built-in camera of the phone. Our sample cassette design contains a hydrophilic membrane filter that is in contact with a waste reservoir made out of absorbent pads, capable of screening >10mL of sample. Giardia cysts in water samples are stained with fluorescein-labeled antibodies specific to Giardia. After incubation for labeling at room temperature, the water sample is filtered through the membrane and the sample cassette is attached to the input-port on our cellphone microscope. The fluorescence signal of stained cysts is imaged over a wide field-of-view (~0.8 cm2) and the acquired image is digitally processed to count the number of cysts on the membrane. This field-portable cellphone-enabled fluorescent microscopy and pathogen detection platform could be useful for screening and quantification of water contamination even in resource scarce environments.

Abstract Title: Using fluid viscoelasticity to expand the stress ranges in single-cell mechanophenotyping

Abstract: Cell mechanical properties are deeply connected to cellular processes and changes in cell state and thus attractive as potential label-free diagnostic markers. Herein, we advance our deformability cytometer (DC) technology, which acquires mechanical properties of thousands of cells per second (Fig. 1), by employing viscoelastic fluids. The DC requires a finely-tuned flow profile in an extensional flow junction to achieve consistent stretching of cells, and thus, only cells with a certain size range (10 ~ 20 µm) have been able to be examined within a certain stress regime (corresponding to ~700 µL/min flow rate). To overcome this limitation, this work utilized fluorescence imaging to monitor movement of fluorescently labeled microbeads in the cross sectional area of the device, and commercially available lambda-DNA to provide the desired viscoelasticity to the fluid. We examined 5 different particle sizes (1 to 30 µm) in the flow rate range of 100 to 1400 µL/min (Re = 15 to 300). The behavior of beads in the junctional area was categorized into five different phases with phase I-III potentially usable for DC. The results indicate that, as the DNA concentration increases, the DC-applicable (phase I-III) flow rate range shifts from Re = 280-300 to Re = 70-300 for 10 µm. The findings in this study will significantly expand the capability of DC in mechanophenotyping, and the advanced DC will facilitate exploration of the impact of different stresses and strain/strain rates on the measured cell mechanical properties.

Abstract Title: Google Glass Based Immunochromatographic Diagnostic Test Analysis

Abstract: Recent advances in wearable consumer electronics such as smart watches and glasses provide unique opportunities for simpler and more integrated designs for biomedical imaging and diagnostics tasks. Here we demonstrate the ability of one such device, the Google Glass, to perform both qualitative and quantitative analysis of lateral-flow based immunochromatographic rapid diagnostic tests (RDTs) through a hands-free voice-controllable software interface. After using voice commands to view and then image one or more RDTs, each labeled with a Quick Response (QR) code (see the Figure), our custom-designed Glass application sends the image and related information (e.g., GPS) to remote servers for digital analysis and evaluation. The evaluated test results are then returned to the originating Glass and also saved and made available via a secure web server in geospatial and tabular representations. We validated the performance of this system by evaluating qualitative (i.e., yes/no) HIV tests and quantitative prostate-specific antigen (PSA) tests. For the PSA tests, we activated and imaged Free and Total PSA tests at concentrations ranging from 0 ng/mL to 200 ng/mL and generated calibration curves mapping the analyzed intensity values against PSA concentration. Providing an easy-to-use digital platform for colorimetric diagnostic tests, our wearable interface can reduce manual test errors from inconsistent training and provide quantitative and automated digitization of test results for real-time spatio-temporal tracking of various diseases and medical conditions.

 Abstract Title: Enhanced Detection of Protein in Urine by Evaporation on a Superhydrophobic Plastic

Abstract: Protein in urine can be detected using a simple colorimetric output by evaporating droplets on a superhydrophobic (SH) surface. Evaporation on a SH surface allows fluid to dramatically concentrate; the weak surface adhesion allows the droplet of fluid to constantly decrease its footprint area and contact diameter. On a SH surface, pure water completely evaporates. Molecules in solution, however, are confined to a footprint 8.5x smaller than the original and are greatly concentrated. This concentrating effect leads to enhanced detection, and by evaporating on a SH surface, protein detection is 16x more sensitive than not evaporating (in the linear detectable range) and is greater than 160x more sensitive than the glass slide control. With the low-cost fabrication method and simple technique, highly sensitive detection can be achieved in a low-cost platform. Utility is demonstrated by detecting protein in urine in the pre-eclampsia range (150-300µgmL-1) for pregnant women. This technique is simple to implement, is relatively fast (1-3hr), and does not require external processing or preparation. The colorimetric signal negates the need of expensive labeling and external equipment, as in fluorescence detection. Importantly, this simple method could also be readily integrated with more advanced detection techniques for improved detection. Finally, these SH surfaces are extremely simple and inexpensive to manufacture for true low-cost diagnostics.

Abstract Title: BioGames: A Game-based Framework for Crowdsourcing Biomedical Image Analysis and Training of Diagnosticians

Abstract: Various approaches over the past decade have tackled difficult and time-consuming image analysis problems by combining human-based crowdsourcing with statistical analysis. Similarly, we have recently created a mathematical framework leveraging crowdsourcing games for biomedical image analysis and diagnosis, and demonstrated its effectiveness for tele-diagnosis of malaria from microscopic images of red blood cells (RBCs) using a minimally trained human crowd. A group of gamers (experts as well as non-experts) used the BioGames platform to collectively generate a set of statistical gold standard labels for individual RBC images, marking them as positive (infected), negative (uninfected), or questionable (the image provides insufficient information for a reliable diagnosis), creating a cell library of over 2850 distinct RBC images, with ~4% and ~5% labeled as positive and questionable, respectively. Using this image library, we have also launched a training game (see the Figure), playable through a web browser, whereby diagnosticians can assess their level of training against their fellow peers. After diagnosing each set of ~500 cells per game, diagnosticians receive a quantified score based on their diagnoses as well as training feedback in the form of labeled images of misdiagnosed cells. We plan to expand our image database/library using the BioGames platform to generate gold labels for new RBC images and make this platform a standard digital tool for malaria diagnostic training. http://biogames.ee.ucla.edu/

 

Abstract Title: Diffusion and control of single particles in pores with combined pressure and dynamic voltage

Abstract: Passage of single molecules and particles through pores is the basis of resistive-pulse sensing. We introduced additional control by modulation of the driving voltage during particle translocations in combination with hydrostatic pressure. Balancing both these forces acting on the particles allowed us to observe diffusion of single particles in the pore for tens of seconds, and quantify their diffusive coefficient. Diffusion coefficients of individual particles was determined based on variance of their local diffusion velocities. This method for measuring diffusion coefficient is applicable to particles of difference sizes, does not require fluorescence labeling, and is entirely based on ion current recordings. Furthermore we were also able to analyze the signal in real time (i.e. remove capacitance, calculate percent signal change), allowing us to switch the voltage whenever the particle began to leave the pore and thus transport the same particle back and forth within the pore. All experiments were performed with charged or uncharged polystyrene particles passing through single 11 µm long pores in a polyethylene terephthalate (PET) film. The pores were prepared by the track-etching technique, which when applied to PET films leads to pores with undulating diameter along the pore axis, as seen in the pulse shape of all translocations. These methods will be especially useful for the analysis of species present in a solution in low concentrations where statistics on an ensemble of particles/molecules must be replaced by statistics based on one particle studied many times.

Abstract Title: Spatio-temporal Mapping of Mercury Contamination using a Smartphone

Abstract: We demonstrate a cost-effective handheld mercury detection platform installed on a smartphone for spatio-temporal mapping of mercury contamination in water samples. This platform integrates an opto-mechanical attachment (< 40 grams) to the camera module of a smartphone to digitally quantify colorimetric transmission signals of a mercury-specific gold nanoparticle (Au NP) assay running on the phone [1]. This Au NP assay displays a distinct color change from red to blue in the presence of mercury ions due to the binding ‘competition’ of a mercury-specific aptamer sequence between mercury ions and positively charged Au NPs. The transmission intensities of this assay are quantified ratiometrically by illuminating the sample tubes with two LEDs at red (523 nm) and green (625 nm) wavelengths simultaneously. A custom-developed Android application was also installed on the smartphone for rapidly processing the acquired images and displaying the test results within less than 7 seconds. The limit of detection of mercury ions was determined to be ~3.5 parts-per-billion (ppb), which is on the same order of magnitude with the US EPA and WHO’s mercury reference concentration in drinking water (2 ppb and 6 ppb, respectively). Spatio-temporal mapping of mercury contamination at over 50 locations in California was also demonstrated.

 

Cell Tissue Organ Engineering

Abstract Title: The effect of scaffold macroporosity on angiogenesis and cell survival in tissue-engineered smooth muscle

Abstract: Angiogenesis and survival of cells within thick scaffolds is a major concern in tissue engineering. The purpose of this study is to increase the survival of intestinal smooth muscle cells (SMCs) in implanted tissue-engineered constructs. We incorporated 250-µm pores in multi-layered, electrospun scaffolds with a macroporosity ranging from 15% to 25% to facilitate angiogenesis. The survival of green fluorescent protein (GFP)-expressing SMCs was evaluated after 2 weeks of implantation. Whereas host cellular infiltration was similar in scaffolds with different macroporosities, blood vessel development increased with increasing macroporosity. Scaffolds with 25% macropores had the most GFP-expressing SMCs, which correlated with the highest degree of angiogenesis over 1 mm away from the outermost layer. The 25% macroporous group exceeded a critical threshold of macropore connectivity, accelerating angiogenesis and improving implanted cell survival in a tissue-engineered smooth muscle construct.

Abstract Title: Consistency of Cardiomyocyte Self-assembly

Abstract: In biology, organization at multiple scales drives functionality. Current advances in staining and imaging of biological tissues provide a wealth of data, but there are few metrics to quantitatively describe these findings. There is a need for a quantitative characterization of consistency of orientation of different biological constructs within a cell. The co-orientational order parameter (COOP), designed based on the mathematical framework of a classical parameter (orientational order parameter), can be used to quantify consistency at multiple scales. Microcontact printing of extracellular matrix was used to engineer various shaped neonatal rat ventricular myocytes. To quantify the orientation of sarcomeres and actin fibers we adapted a previous MATLAB code. Then we used a custom MATLAB code to calculate the COOP between pairs of cells for both sarcomeres and actin. To test consistency of cytoskeleton architecture within patterned cardiomyocytes, we compared actin and sarcomere distributions across five triangular cells. Qualitatively cardiomyocytes constrained to a triangular extracellular matrix island have consistent architecture with myofibril bundles spanning the edges of the cell. However, our results indicate that the architecture is not fully consistent on the length scale of a few sarcomeres. We have used the COOP to show that cardiomyocyte self-assembly is only consistent at some length scales but not others. In the future, the COOP can be used to identify length scales at which cells of different origin are consistent in the organization of their cytoskeleton.

Abstract Title: Cell-in-Gel system imposes mechanical load on single intact cardiomyocytes

Abstract: Objective: Increased mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause arrhythmias and heart failure. However, little is known about the mechano-transduction mechanisms that underlie heart disease development due to previous lack of techniques to control mechanical stress on intact cardiomyocytes necessary for investigating at cellular and molecular levels. Here we use a system to study the mechanical load effects on modulating myocyte Ca2+ signaling and contraction dynamics. Methods: We developed a novel Cell-in-Gel system that controls mechanical load on single rabbit myocytes during excitation-contraction coupling in a 3D elastic gel matrix composed of polyvinyl alcohol and a crosslinker. Myocyte contraction and Ca2+ transients were measured in-gel and compared with load-free cells. Results: Contracting cells in-gel showed significantly lower fractional shortening (13.5 ± 0.4% in-gel, 14.7 ± 0.3% load-free), exhibiting an 8% decrease. However, the systolic Ca2+ transient was greater in-gel (fluorescence ratio peak 1.47 ± 0.07 in-gel, 0.85 ± 0.03 load-free), revealing mechano-chemo transduction translating external stress to intracellular Ca2+ increase. Conclusions: Our Cell-in-Gel system provides a versatile experimental method to control mechanical stress for investigating mechano-chemo transduction pathways in intact myocytes. These experimental results are consistent with our mathematical modeling predictions, demonstrating the mechanical load effects on altering myocyte Ca2+ handling and contraction dynamics.

Abstract Title: Site Directed Differentiation of Embyronic Stem Cells Using Insoluble VEGF in Fibronectin

Abstract: Fibronectin (Fn) has been identified as containing a binding site for vascular endothelial growth factor (VEGF), a biochemical noted for is potent mitogenic and angiogenic affects on endothelial cells (EC), as well as, it role in vascular cell fate. Therefore, we examined the role of insoluble VEGF in directing EC fate from embryonic stem cells (ESC). Using our stage-specific serum-free medium formulation, we examined the potency of insoluble VEGF, soluble VEGF, as well as, combined insoluble and soluble VEGF on the initial stage of induction of Flk-1+ vascular progenitor cells. Initial studies suggest that the insoluble VEGF (VEGF-Fn) yields a 90% differentiation rate compared with 75% differentiation rate using Fn with soluble VEGF. This data confirms that less soluble VEGF is required for vascular induction of ESC. Moreover, this technique can be used to incorporate spatial patterning for directing EC fate into appropriate vascular structures for vascular tissue engineering.

Abstract Title: Effects of Substrate Stiffness on Reprogramming from Fibroblasts to Neurons

Abstract: Direct reprogramming is the conversion of one cell type into a completely different cell type. In 2010, Vierbuchen et al successfully created induced neurons via direct reprogramming from mouse embryonic fibroblasts and post-natal tail-tip fibroblasts by the forced expression of three transcription factors Brn2, AsCl1 and Myt1L (BAM in short) with an efficiency of 1.8-7.7%. On a separate note, it has long been known that substrate stiffness can have an effect on cell adhesion, proliferation, motility and even differentiation potential of various adult stem cells. However, there has yet to be any reports on the effect of substrate stiffness on the reprogramming process. Here, we investigate the effects of modulating substrate stiffness on direct reprogramming of adult mouse fibroblasts into functional neurons. We observed that direct reprogramming from fibroblasts to induced neurons is favored on the intermediate stiffness gel. The neurons formed were mature and functional. Further studies will provide insights into the mechanism through which substrate stiffness affects this reprogramming process and increase our understanding of the effects of substrate stiffness on genetic and epigenetic mechanisms that determine cell fate. Successfully increasing the reprogramming efficiency will prove to be valuable with potential applications in the creation of disease-specific models for drug discovery

Abstract Title: Activation of CD11c primes foamy monocytes for recruitment on VCAM-1 under shear

Abstract: Recent reports have shown that foamy macrophages in atherosclerotic plaque are derived from monocytes recruited from blood during hypercholesterolemia. However, the phenotype of the recruited monocytes and the mechanism through which lipid uptake drives their recruitment remains unclear. Our previous studies show that ß2-integrin CD11c is upregulated on the monocyte during hyperlipidemia and cooperates with ß1-integrin very late antigen-4 (VLA-4) to capture monocytes on endothelium expressing vascular cell adhesion molecule-1 (VCAM-1) under physiological shear. This led to the hypothesis that activation of CD11c on foamy monocytes in blood increases VLA-4 function (affinity and avidity) via formation of focal adhesion complexes that facilitate capture on VCAM-1 under shear. To examine the effects of hyperlipidemia on adhesion molecule expression we fed apoE-/- mice a high fat diet for 1-5 weeks. CD11c expression doubled on foamy monocytes at 1 week on HFD. Since the integrins CD11c and VLA-4 support monocyte recruitment on VCAM-1, we sheared mouse whole blood in our microfluidic flow channels over recombinant VCAM-1. We detected a ~30% increase in enrichment of foamy monocytes after 1 week HFD. Using in plane total internal reflected fluorescence (TIRF) to image bound adhesion molecules on recombinant VCAM-1, we determined that CD11c and VLA-4 colocalize at the contact site to support arrest during hypertriglyceridemia. Immunoprecipitation of CD11c revealed that VLA-4, spleen tyrosine kinase (Syk), and paxillin formed focal adhesion complexes following activation of CD11c with lipid or allosteric binding antibody that stabilizes CD11c into high affinity conformation on monocytes in suspension. We attribute the increase in efficiency of capture to activation of CD11c and formation of focal adhesion complexes with VLA-4 via a Syk and paxillin dependent mechanism.

Abstract Title: Reconfigurable microfluidics with integrated aptasensors for monitoring intercellular communication

Abstract: We report the development of a microsystem integrating anti-TNF-a aptasensors with vacuum-actuatable microfluidic devices that may be used to monitor intercellular communications. Actuatable chambers were used to expose to mitogen a group of ~600 cells while not stimulating another group of monocytes only 600 µm away. Co-localizing groups of cells with miniature 300 µm diameter aptamer-modified electrodes enabled monitoring of TNF-a release from each group independently. The microsystem allowed observation of the sequence of events that included 1) mitogenic activation of the first group of monocytes to produce TNF-a, 2) diffusion of TNF-a to the location of the second group of cells and 3) activation of the second group of cells resulting in the production of TNF-a by these cells. Thus, we were able to experimentally verify reciprocal paracrine crosstalk between the two groups of cells secreting the same signalling molecule. Given the prevalence of such cellular communications during injury, cancer or immune response and the dearth of available monitoring techniques, the microsystem described here is envisioned to have significant impact on cell biology.

Abstract Title: STRETCHING CARDIAC TISSUE: TOWARDS EX VIVO STUDIES ON STEM CELL DIFFERENTIATION, INTEGRATION, AND REPAIR

Abstract: Cardiovascular diseases have long been the leading cause of death in the United States. This fact has prompted a great deal of research into cardiac physiology, pathology, and development of experimental and/or therapeutic devices. In order to understand the underlying mechanisms of the various cardiovascular diseases and to assess long-term experimental therapeutics, it is vital to develop a functionally intact explant model of beating heart tissue for these studies. Currently, vital organotypic human heart can be maintained at liquid-air interface, with some loss of structural integrity, for up to 1 month. In order to extend both functionality and tissue viability for up to 3 months, we have designed and built a tissue-stretching device for preservation and maintenance of these organotypic cardiac tissue slices. Our device has been designed to 1) anchor tissue preparations with minimal tissue destruction, 2) fully embed both sides of tissue into cell culture medium, 3) provide mechanical stimulation/stretching with 0.5 – 2Hz frequency and strain at 5% – 15% elongation, while also maintaining sterility of culture environment. We expect that this stretching device to aid longer term viability and functional physiology of the cardiac tissue explants, as well as, enable unique studies on cell/tissue integration with heart explants.

 

Abstract Title: Decoupling the Influence of Fluidic Agitation and Aggregate Size on Human Pluripotent Stem Cells in Dynamic Suspension

Abstract: Despite the potential of human pluripotent stem cells (hPSCs) as an ideal cell source for future cell therapy applications, a robust scalable culture system that can produce sufficient number of clinical stem cell products is currently lacking. Dynamic suspension culture is a promising platform because it is easily scalable and automated, thereby reducing cost of cell production. However, propagation of hPSCs in dynamic suspension has not been explored until recently, and the associated microenvironmental factors that regulate fate decision of hPSCs are poorly understood. In particular, fluidic agitation is unique to dynamic suspension and can play an important role. We assessed impacts of different agitation rates (0-100 rpm) on hPSC proliferation and maintenance of pluripotency by using a spinner flask. After 7 days of dynamic suspension culture, moderate agitation at 60 rpm achieved the highest cell yield (53 fold increase) while maintaining high expression of pluripotent markers. This condition also produced the most uniformly sized cell aggregates with 200-300 µm in diameter, whereas other agitation rates resulted in broader size distribution. This result presented a relationship between size of cell aggregates and the cell yield, indicating an optimal aggregate size for survival and growth of hPSCs. To confirm this observation, we cultured hPSCs aggregates of prescribed sizes (100-500 µm) in mTeSR under static condition for 7 days. Overall, aggregate size at 300 µm had the highest cell yield (40 fold increase) and viability (90%) as well as high expression of pluripotency makers. Sizes below or above 300 µm displayed a decrease in both cell yield and viability. Particularly, larger sizes (>400 µm) resulted in early germ layer differentiation. Collectively, these studies shows that aggregate size is a critical parameter through which fluidic agitation, by modulating aggregation kinetics, can affect fate decisions of hPSCs in dynamic suspension.

Abstract Title: Shear Stress-Activated Wnt-Angiopoietin-2 Signaling Recapitulated Vascular Repair in Zebrafish Embryos

Abstract: Fluid shear stress intimately regulates vasculogenesis and endothelial homeostasis. The canonical Wnt/β-catenin signaling pathways play an important role in differentiation and proliferation. In this study, we investigated whether shear stress activated Angiopoietin-2 (Ang-2) via the canonical Wnt signaling pathway with an implication in vascular endothelial repair.

Oscillatory shear stress (0 ± 3 dyn/cm2 at 1 Hz) up-regulated both TOPflash Wnt reporter activities (2.35±0.46, p < 0.05 vs. control, n=4) and Ang-2 RNA and protein expression in human aortic endothelial cells (HAEC) accompanied by an increase in nuclear β-catenin intensity. OSS-induced Ang-2 and Axin-2 mRNA expression was down-regulated in the presence of a Wnt inhibitor, IWR-1, but was up-regulated in the presence of a Wnt agonist, LiCl. Ang-2 expression was further down-regulated in response to a Wnt signaling inhibitor, DKK-1, but was up-regulated by Wnt agonist Wnt3a. Both DKK-1 and Ang-2 siRNA inhibited endothelial cell migration and tube formation, which were rescued by human recombinant Ang-2. Both Ang-2 and Axin-2 mRNA down-regulation was recapitulated in the heat-shock inducible transgenic Tg(hsp70l:dkk1-GFP) zebrafish embryos at 72 hours post fertilization (hpf). Ang-2 morpholino injection of Tg (kdrl:GFP) fishimpaired subintestinal vessel (SIV) formation at 72hpf, which was rescued by zebrafish Ang-2 mRNA (zAng-2) co-injection. Inhibition of Wnt signaling with IWR-1 also down-regulated Ang-2 and Axin-2 expression, and impaired vascular repair after tail amputation, which was rescued by zAng-2 injection.

Shear stress activated Ang-2 via canonical Wnt signaling in vascular endothelial cells, and Wnt-Ang-2 signaling is recapitulated in zebrafish embryos with a translational implication in vascular development and repair. 

Abstract Title: Cell Mechanical Phenotyping for Screening microRNA-based Therapeutics

Abstract: Mechanical changes at the single cell level provide important insights into the metastatic potential and drug sensitivity of cancer cells. For example, recent findings reveal that highly invasive cancer cells are more compliant (or ‘softer’) than less invasive cancer cell lines. The mechanical properties of metastasizing cells could have a functional role in disease progression. Known regulators of cell mechanical phenotype include cytoskeletal proteins such as actin and microtubules. Lamin proteins are major determinants of nuclear shape/stability and influence the ability of migrating cells to maneuver through extracellular matrix. The discovery that microRNAs (miRNAs) regulate pathways that orchestrate these phenotypic changes presents an exciting therapeutic opportunity: controlling miRNA levels can revert malignant phenotypes. Using a microfluidic assay to probe cell deformability, our studies reveal that upregulating levels of miR-130b, miR-508-3p, and miR-509-3p makes human ovarian cancer less deformable; in vitro scratch wound assays also reveal that treated cells are less invasive. Taken together with immunofluorescent imaging of subcellular structures and quantitative polymerase chain reaction (qPCR) to quantify expression levels of key structural proteins, this method provides a new approach to study the role of cellular and nuclear deformability in the metastatic progression of ovarian cancer.

Abstract Title: Motoneuron- and signal-mediated maturation of hiPSC-derived skeletal myospheres

Abstract: The availability of human embryonic stem cells (hESC)- or human induced pluripotent stem cells (hiPSC)-derived organs and tissues provides the unique opportunity to recapitulate salient features of human diseases in a dish model that can be exploited to understand their molecular pathogenesis and to identify target for therapeutic interventions. Unfortunately, generation of in dish models of human skeletal muscles has been prevented so far by the “intrinsic resistance” of hESCs to differentiate into muscle. Our recent discovery, that the absence in hESCs of the SWI/SNF component BAF60C (encoded by SMARCD3) prevents the activation of the myogenic program in hESCs, overrode this bottleneck and inspired a protocol for the generation of hESC and hiPSC-derived three-dimensional (3D) skeletal myospheres composed of myofibers. One limitation of this protocol relates to the incomplete maturation of myospheres, as evidenced by the lack of unidirectional organization and alignment of myofibers, irregular formation laminin and dispersed localization of Pax7-expressing, putative satellite cells. Moreover, although spontaneous contractility can be observed in skeletal myospheres, contractile events occur randomly and cannot be deliberately evoked. To improve the differentiation of the myospheres and proper localization of muscle satellite cells (MuSCs), we are employing a bioengineering approach consisting of co-encapsulation of the embyoid bodies and motor neurons at early stages of differentiation (day5) in a synthetic hydrogel that simulates the physiological stiffness provided by extracellular matrix and is predicted to promote laminin organization, myofibers alignment, contraction through NMJs, and proper localization of MuSCs. Overall, development of 3D-innervated myospheres from hiPSC will provide a unique tool to understand fundamental principles of muscle development and to develop patient-derived in dish models of neuromuscular diseases.

Abstract Title: Tracking and quantifying focal adhesion kinase and paxillin at lamellipodial protrusion in migrating endothelial cells

Abstract: Focal adhesion kinase (FAK) and paxillin are involved in focal adhesion (FA) disassembly and formation, which can regulate cell migration, intracellular signaling, and remodeling of actin filaments. We used time-lapse double-color imaging to monitor concurrently the spatial-temporal dynamics of FAK and paxillin in live endothelial cells (ECs) co-transfected with GFP-FAK and DsRed-paxillin, and the fluorescence intensities (FI) of paxillin and FAK at FAs were determined in cell front, center and rear. The mean FAK/Paxillin FI ratio is highest at cell front (mean±SEM: 4.73±0.48), near 1 at cell center (0.95±0.11), and lowest at cell rear (0.64±0.05) (P<0.01). Determination of the time difference between the assemblies of FAK and paxillin at FAs in lamellipodial protrusion (LP) showed that FAK assembly occurs ahead of that of paxillin at individual FAs. Computational analysis indicates that FAK reaches its maximum intensity earlier than paxillin by about 4 min. The assembly of FAK is at LP ahead of paxillin Indicates that, while both molecules play important roles in modulating FA dynamics during cell migration, FAK promotes FA formation ahead of paxillin at the cell leading edge. This work was supported by NHLBI Research Grants HL-104402 and HL-106579 (S.C.).

Abstract Title: Disturbed Flow Induces Oxidative Stress Mediated Autophagy

Abstract: Temporal and spatial variations in shear stress are intimately linked with vascular metabolic effects. Autophagy is a tightly regulated intracellular bulk degradation/recycling system for maintaining cellular homeostasis. We postulated that disturbed flow promoted autophagy with an implication in mitigating mitochondrial superoxide production. In the lesser curvature of human aortic arch where atherogenic flow or oscillatory shear stress (OSS) occurred, we observed a paucity of p62 staining indicative of increased autophagy activity. Whereas in the greater curvature where atheroprotective or pulsatile shear stress (PSS) developed, p62 was prominent. While in vitro, OSS significantly increased autophagy as well, as measured by increased LC3-II/LC3-I ratios and GFP-LC3 dots/cell in human aortic endothelial cells (HAECs). The OSS-induced autophagy was attenuated with adenovirus MnSOD, antioxidant N-acetylcysteine, or inhibition of c-Jun N2-terminal Kinase (JNK). These findings demonstrate that disturbed flow preferentially induces autophagy by oxidative stress and JNK signaling with an implication in maintaining endothelial homeostasis.

Abstract Title: The effect of cell shape on macrophage phenotype

Abstract: As central orchestrators of the immune system, macrophages fulfill their diverse functional roles by assuming a spectrum of functional phenotypes. In the presence of dangerous foreign substances, macrophages can acquire the pro-inflammatory phenotype (M1) and release cytotoxic mediators to fight infection. On the other hand, in a wound healing environment, macrophages can also polarize toward a pro-healing phenotype (M2) and assist in tissue repair. Understanding the microenvironmental cues that regulate macrophage phenotype has significant clinical implications as aberrant macrophages polarization has been linked to many diseases. Despite much effort in elucidating the role of soluble factors such as cytokines in macrophage polarization, how physical and mechanical signals may influence macrophage phenotype remains unclear. In particular, the effect of cell shape on macrophage polarization has not been explored, even though macrophages are known to adopt different cell morphologies in vivo. We, and others, have previously observed that macrophages polarized towards M1 and M2 phenotypes using cytokines assume dramatically different cell morphologies: M2-polarized cells exhibited a significantly higher degree of cell elongation than M1 cells. In this work, we use a micropatterning approach to directly control shape and show that elongated cell shape, independent of soluble factors, can promote macrophage polarization towards an M2 phenotype.

Abstract Title: Massively parallel magnetic particle manipulation system for exploring intracellular force response

Abstract: Gaining statistically relevant models of cellular mechanisms remains an essential step in progressing biomedical research. Particularly, the study of cellular force response continues to be heavily explored. Indeed increasing understanding of how cells interpret and respond to internal and external forces can inform development of treatment approaches and therapeutics. Traditionally, these studies are carried out using micromanipulation tools such as optical tweezers. However, these tools face inherent challenges with biocompatibility and experimental throughput. To address these challenges, we have developed a massively parallelized magnetic tweezer platform which can impart pN scale forces on superparamagnetic nanoparticles (SPIONs). The system consists of a joystick controlled robot which drives the orientation of a bulk magnetic field, a microarray of permalloy pillars, and fluorescently labeled SPIONs. When the bulk field is brought into registration with the pillar microarray, the pillars magnetize in alignment with the bulk field inducing forces on the SPIONs directed towards the field maxima. Dynamic forcing can be achieved by cycling the bulk field thereby manipulating particles across the pillar microarray. We are using this system to explore cellular response to localized and dynamic cytoplasmic forcing through SPIONs internalized into HeLa cells. Upon application of oscillatory forcing at 0.003Hz, we observed rapid filopodial contraction within 15 minutes after force application. We are exploring this phenomenon further in the context of cellular force response.

 

Abstract Title: Linear Regression Analysis of Combinatorial Parameters for Endothelial Cell Fate Optimization

Abstract: Embryonic stem cells (ESC), including induced-pluripotent stem (iPS) cells can be differentiated into functional endothelial cells (EC) in vitro by embryoid body or monolayer culture methods of induction. Regardless of the methodology, directing cell fate of pluripotent stem cell remains an inefficient process. This study examined multiple potential parameters for optimization of EC fate from two ESC human lines (H7 and H9) and 4 mouse ESC lines (including E14 and R1, as well as, in-house derived ESC lines, A3 and B2). Using our staged induction methodology combined with sera-free medium formulations, the factors directing the expression of Flk-1/KDR and VE-cadherin positive cells were examined for the first and second stages of differentiation, respectively. Parameters examined included: seeding density, matrix, VEGF, and bFGF concentrations. After generating large data sets on various combinations and levels of these parameters, data was analyzed using linear regression modeling in R. Categorizing parameters as either continuous for discrete factors, the statistical output suggests that both human and mouse ESC differentiation of EC was most significant with high 50ng/ml bFGF (p<0.05) without the addition VEGF. However, some cell lines, A3 and B2, were more responsive to 10ng/ml VEGF with no bFGF. Fibronectin at 50ug/ml was a significant factor (p<<0.001) in directing EC fate from all lines of human and mouse ESC examined. Most interestingly, a high cell seeding density, 10,000 cells/cm2, was the most important factor in generating the greatest Flk-1/KDR vascular progenitor cells at stage 1. These findings suggest that hypoxia, cell-to-cell signaling, matrix signaling, and/or bFGF signaling play greater roles in EC fate compared with VEGF signaling.

Abstract Title: Optical Tweezers Studies To Investigate The Role Of Furin Processing Of Notch

Abstract: Notch signaling pathway is a critical juxtacrine signaling pathway necessary for cell fate determination during development of most metazoa. The pathway receptor is trans-activated by the ligand on the apposing cell by pulling force generated by endocytosis leading to destabilization of a structure called Negative Regulatory Region (NRR) on the Notch Extracellular domain (NECD) resulting in regulated intramembrane proteolysis by ADAM 10 and ?-Secretase proteases and NECD shedding. Putative Notch receptor is expressed on the cell surface as a heterodimer after cellular processing during maturation by a convertase called Furin (S1 cleavage) and its role in NRR disengagement and NECD shedding is not yet understood. We propose to investigate the role of S1 cleavage using optical tweezers based dynamic force spectroscopy of mammalian Notch -Delta-like-ligand interaction. Protein-A microbeads coated with Delta-like-ligand-1 fused with IgG Fc (D1Fc) were used in touch and release experiments on Notch1 (N1) expressing L cells, and that expressing mutant Notch1, with S1 cleavage site deleted (N1DFC) , on our custom built optical tweezers system. . Probability density of several such interaction forces (bond ruptures) was obtained, and Force-Extension curves for each such specific rupture force were analyzed. Force spectra reveal several modes indicating three possible rupture points after N1-D1Fc binding indicative of rupture due to S1 cleavage, heterodimer breakage or N1-Dll-1 bond breakage. However, the force spectra after DFCN1-Dll-1 showed indistinctive rupture points. . Mapping of rupture forces to their corresponding extension and pathlength (path of the force-extension curve till the rupture) showed two distinct 3D modes unique to the N1-Dll-1 interaction while DFCN1-Dll-1 showed only one. Surprisingly, this single 3D mode of DFCN1 extension overlapped with one of the modes of N1 indicating that the mutant DFCN1 is restricted to one type of activation biophysics.

Abstract Title: Investigating the roles of S1P and hypoxia on mature and progenitor endothelial cells

Abstract: Therapeutic angiogenesis involves the delivery of prosurvival factors or blood vessel forming cells to stimulate neovessel formation within hypoxic tissue and restore oxygen-rich blood perfusion. Our work pursues the promising idea of locally delivering sphingosine-1-phosphate (S1P) with spatiotemporal control to efficiently recruit outgrowth endothelial cells (OECs), a subset of endothelial progenitor cells, from circulation and promote revascularization in ischemic tissue. S1P is a bioactive lysophospholipid that augments endothelial cell (EC) proliferation, migration, apoptosis, and differentiation to regulate vascular homeostasis. Both hypoxic stress and local S1P concentration modulate the S1P receptor (S1PR1-5) profile on the surface of ECs and are thus hypothesized to influence the overall response to S1P presence. Vascular endothelial growth factor (VEGF) is a critical factor in orchestrating angiogenesis and regulating vascular permeability. Here, we addressed the hypothesis that an interaction exists between S1P, VEGF, and hypoxic signaling on the angiogenic response of both microvascular ECs and OECs. Co-stimulation with S1P and VEGF resulted in significantly greater angiogenic activity as assessed by proliferation, motility, directed migration/matrix invasion, and 3D sprouting under both normoxia and hypoxia. Additionally, S1P differentially enhanced greater sprouting formation than VEGF did under hypoxia. This suggests that hypoxia augments the interplay between S1P and VEGF signaling on angiogenic activity. Furthermore, S1P was incorporated within an alginate hydrogel and the release profile was determined over the course of one month in order to evaluate the ability to use this delivery system for therapeutic angiogenesis in vivo in the future. Overall, the results of these studies support the notion that S1P holds promise as a therapeutic agent for vascular repair in ischemic tissue.

Abstract Title: Role for Stiffness in Vascular Fate

Abstract: Embryonic stem cells (ESC) have been explored as tools for studying development, as well as, potential sources for a large number of therapies in regenerative medicine. Traditionally, ESC are cultured on tissue culture plastic, however; it has been recently shown that the stiffness of the environmental substrate can direct the cells towards various cell lineages. Our laboratory is specifically interested in examining the combined roles of biochemical and physical signaling in cardiac and vascular cell fate and patterning these vascular cells into vascular branch-like tress. Using our novel mouse ESC that expresses a GFP reporter under the Tie-2 and an RFP reporter under alpha smooth muscle actin and serum-free induction mediums, we examined the role of stiffness in the diverging fate of Flk-1+ vascular progenitor cells. The results indicate that both of the Flk-1+ vascular progenitor cells and human umbilical vein endothelial cells (HUVEC) preferentially adhere to 10 kPa compared with the 1 kPa, and 34 kPa compared with the 10 kPa. We also observed both the GFP/Tie-2+ endothelial-like and RFP+ smooth muscle-like cells outgrowths from the Flk-1+ cells, with the GFP/Tie-2+ cells dominating the cultures, supporting the role of stiffness in vascular fate. Next, we generated a vascular fractal-like pattern reverse mold and have stamped fibronectin vascular pattern onto non-tissue culture treated plastic. Using these combined technologies, we have been able to generate vascular branching patterns with our ESC-derived EC.

Abstract Title: Quantifying change in stiffness heterogeneity in 3D around cells embedded in natural ECMs

Abstract: Cells in their natural microenvironment interact and respond to mechanical stimuli generally conveyed by or through the extracellular matrix (ECM), the molecular yet mechanical scaffolding of natural tissues. Cells transduce mechanical cues into molecular signal and responses through a process known as mechanotransduction, which plays an indispensable role in influencing critical behaviors such as proliferation, migration, differentiation etc. As part of mechanotransduction, cells partake in a sensitive force-balance with their surrounding matrix in a process called mechanoreciprocity. This is a dynamic process in which the local ECM fiber network is deformed and remodeled with matrix metalloproteinases and cell-generated contractile forces. Measuring the change in stiffness over time as a cell probes and deforms its surrounding microenvironment is a nontrivial task that would provide greater insight into these processes. Our new system, automated optical tweezers active microrheology (aAMR), allows us to measure change in stiffness around cells in real time. For the first time, we are able to quantitatively map stiffness changes as a result of cellular behavior. Unlike traction force microscopy, which is only useful in synthetic elastomers and atomic force microscopy which lacks axial resolution, aAMR can probe deep within a 3D gel. By looking at the stiffness change between cells and their surrounding microenvironment in 3D, we can begin to understand how the control mechanisms behind behaviors like migration and differentiation in ways that were not previously possible.
Abstract Title: Control of pluripotent stem cell colony morphology to enhance lineage specific differentiation

Abstract: Despite their significant therapeutic potential, induced pluripotent stem cells (iPSCs) pose critical risks such as tumorigenesis and teratoma formation in the body. It is suggested that pre-differentiation to target cell types prior to clinical application would reduce such risks. In this context, we have shown that colony morphologies of iPSCs developed by different mechanical microenvironments significantly affects their differentiation potential. In this study, we show that differences in colony morphology are mainly due to intracellular mechanics which direct stem cell differentiation. A human iPSC line was used for guiding differentiation to mesendodermal phenotype using a defined media. Nanofibrous scaffolds with varying mechanical properties were subjected to Collagen Type I surface conjugation to eliminate polymer chemistry for cell attachment. iPSCs were seeded onto scaffolds and pre-cultured in proliferation media with (+RI) or without ROCK inhibitor (-RI). After pre-culture cell colony morphology was visualized using immunocytochemistry. Following differentiation, gene expression was analyzed using real time PCR. On softer scaffolds, iPSCs form round, 3D colonies during pre-culture (-RI) while cells on stiffer scaffolds proliferate in flat, 2D colonies. Cells maintained +RI showed flat colony morphology regardless of stiffness. iPSCs subjected to differentiation, after -RI pre-culture, exhibited a positive relationship between gene expression of mesendodermal markers and scaffold stiffness. However, such correlation was demolished upon the application of RI during pre-culture, which suppressed substrate-stiffness dependent colony morphology development. We have shown that changes in cell colony morphology can mediate efficiency of differentiation towards mesendodermal lineage. Such relationship was inhibited by disrupting intracellular tension, suggesting that interactions with adjacent cells in iPSC colonies significantly affect differentiation.

Abstract Title: ENHANCED ENDOTHELIAL CELL RESPONSE ON SUB –MICROMETER PATTERNED TITANIUM

Abstract: Titanium (Ti) is of particular interest as a biomaterial due to its potential use in pro-healing vascular stents. To remain effective over an extended period, vascular stents must promote surface re-endothelialization. The native vascular endothelium is anchored onto a nanofibrous basement membrane scaffold, with the scaffold topography believed to strongly regulate cell behavior. Thus, to enhance the potential for stent re-endothelialization, we sought to mimic the topographical features of native endothelial basement membrane by fabricating micro- and nano- patterned topography on Ti biomaterial. Using our novel Ti deep reactive ion etching (Ti DRIE) technique, we here report successful fabrication of surface gratings with groove widths down to 500 nm. This is the smallest feature size achieved to date on a bare metal. Next, we evaluated the behavior of cultured human endothelial cells (HEC) cultured on these Ti substrates. We observe that HEC proliferation on these substrates increases considerably with decreasing feature sizes (p-value=0.001 for HECs cultured on 500 nm when compared to those cultured on unpatterned substrates). Notably, response on all patterns is typically greater than the unpatterned control. Importantly, this preferential cell proliferation on sub-micron grooves correlates with more favorable cellular morphology and cytoskeletal structure, as evidenced by significant elongation and alignment along the grating axis. These preliminary findings of endothelial cell response on patterned Ti surface are consistent with previous reports of cell behavior on patterned polymeric substrates, thus underscoring the usefulness and fidelity of our unique Ti DRIE fabrication technique. Work is underway to obtain a deeper understanding of the effects of sub-micron patterned Ti substrates on endothelial cells as it has important implications for the development effective vascular stents and cardiovascular implants.

Systems biology, neurobiology, synthetic biology in Engineering

Abstract Title: Significance of ventricular trabeculation to support ventricular wall with increasing blood flow

Abstract: Maturation of cardiac development requires the delicate morphological formation of ventricular trabeculation. Trabeculae start invaginating into the inner ventricular wall at the end of cardiac looping. However, the mechanical importance of trabeculation is still unclear. Here, we hypothesized that the formation of ridges along the inner ventricle might help to reduce mechanical stress on the ventricle during ventricular filling with cardiac maturation. Zebrafish is unique model to study cardiac development due to their rapid development and optically clear bodies during early embryonic development. To suppress the ventricular trabeculation, we treated Tg(cmlc2:GFP;gata1:DsRed) double transgenic fish with pharmacological inhibitor AG1478. Selective Plane Illuminated Microscopy (SPIM) technique provided 2D fluid domain for simulating mechanical properties for ventricular wall. Digital Particle Image Velocimetry (DPIV) was introduced to measure the inlet velocity from atrium to ventricle. At 5 days-post-fertilization (dpf), wall shear stress (WSS) in control is 20% less than that of AG1478 treated group. Furthermore, the ventricular wall of control group had less percentage of damage under fatigue test due to the thicker wall. At 10 dpf, trabeculation of control group was denser with a thicker wall. While, AG1478 treated fish did not have a change in wall thickness, which led to higher fatigue levels. Thus, our results suggested that ventricular trabculation is necessary to structurally support the ventricle to increasing blood flow against the cardiac wall in a mature heart.

Abstract Title: Electrocorticogram encoding of upper extremity movement duration

Abstract: Electrocorticogram (ECoG) is a promising long-term signal acquisition platform for brain-computer interface (BCI) systems such as upper extremity prostheses. Several studies have demonstrated decoding of arm and finger trajectories from ECoG high-gamma band (80-160 Hz) signals. In this study, we systematically vary the velocity of three elementary movement types (pincer grasp, elbow and shoulder flexion/extension) to test whether the high-gamma band encodes for the entirety of the movements, or merely the movement onset. To this end, linear regression models were created for the durations and amplitudes of high-gamma power bursts and velocity deflections. One subject with 8-by-8 high-density ECoG grid (4 mm center-to-center electrode spacing) participated in the experiment. The results of the regression models indicated that the power burst durations varied directly with the movement durations (e.g. R^2=0.71 and slope=1.0 s/s for elbow). The persistence of power bursts for the duration of the movement suggests that the primary motor cortex (M1) is likely active for the entire duration of a movement, instead of providing a marker for the movement onset. On the other hand, the amplitudes were less co-varied. Furthermore, the electrodes of maximum R^2 conformed to somatotopic arrangement of the brain. Also, electrodes responsible for flexion and extension movements could be resolved on the high-density grid. In summary, these findings suggest that M1 may be directly responsible for activating the individual muscle motor units, and future BCI may be able to utilize them for better control of prostheses.

Abstract Title: Identification and Regulation of Novel Cellulases within Anaerobic Gut Fungi

Abstract: The economical breakdown of plant biomass into simple sugars remains a significant bottleneck in the production of renewable fuels via microbial fermentation. Existing technologies, however, are insufficient due to the recalcitrance of lignin-rich biomass, and the high cost/poor performance of known cellulolytic enzymes. Therefore, there is a critical need to develop new technologies to break down crude biomass into fermentable sugars. Towards this goal, much can be learned by studying how anaerobic gut fungi depolymerize lignocellulose in biomass-rich environments. Anaerobic gut fungi are native to the gut and rumen of large herbivores, where they have evolved unique abilities to break down lignocellulosic biomass through invasive growth and the secretion of powerful enzymes and enzyme complexes (cellulosomes). Towards engineering gut fungi as novel platform organisms for biofuel production, we have isolated a panel of unique specimens and are developing bioinformatics pipelines that have characterized the biomass-degrading machinery of a novel Piromyces isolate from transcriptomic and proteomic studies. Each isolate thrives on lignocellulosic substrates, and secretes multi-protein cellulosome complexes of cellulases, hemicellulases, and cellulose binding domains that can be elucidated by sequencing. To determine the basic metabolic networks that govern biomass hydrolysis within anaerobic fungi, we are employing RNAseq to quantify transcript abundance when simple sugars repress lignocellulosic degradation. Parallel proteomics efforts are underway to compare the regulation of secreted fungal enzymes with those regulated at the transcriptional level. Through these efforts, we will determine how important enzyme groups are coordinated during biomass breakdown across fungal genera. Collectively, this information will establish the molecular framework for anaerobic fungal hydrolysis, and will guide in the development of lignocellulosic biofuels.

Abstract Title: Kepler Workflows for Data and Model Integration in Multi-Scale Systems Biology

Abstract: Integrative multi-scale modeling of complex genotype to phenotype relationships now frequently requires the integration of data, algorithms and software tools from multiple sources, which can significantly complicate efficiency, reproducibility and provenance tracking. Kepler provides an open source environment to integrate these disparate packages. Here we present examples of the use of Kepler for workflows requiring the integration of genomic data, gene-ontology analysis and across-dataset analysis, as well as the combination of clinical data and multi-scale patient-specific models. The automated bioinformatics workflow utilized either online or local microarray data sets and integrated external tools such as Bioconductor packages, AltAnalyze, a python-based open source tool and R-based comparison tool. The automated patient-specific analysis workflow integrated patient-specific clinical electrocardiograms and external tools such as Continuity, Mat lab, and Python. Both automated workflow improved process automation, standardization, objectivity, accuracy, reproducibility, and throughput capacity, thereby advanced intra- and inter- dataset meta analyses of genomic data as well as the feasibility of patient-specific models as useful clinical tools for diagnosis and therapy planning.

Abstract Title: On Intrinsic Dimensionality of Extracellular Action Potentials

Abstract: Linear approaches to low-dimensional feature extraction may not be appropriate when statistical data are generated by a nonlinear interaction of parameters. Equally inadequate are linear methods for determining the dimension of the feature space. This study estimates the intrinsic dimension of extracellular action potentials (EAPs), which can be viewed as the minimum number of nonlinearly interacting parameters sufficient to describe the data. When combined with nonlinear feature extraction methods, this information may lead to a more faithful, low-dimensional EAP representation. These points are demonstrated using EAPs recorded experimentally by a multisensor electrode.

Abstract Title: Magnetic Manipulation with Customizable Bioactive Superparamagnetic Nanoparticles

Abstract: Traditional cellular behavior studies often include the use of mutant generation, gene transfection, and large-scale controlled stimuli. While these techniques are useful in their own right, spatiotemporal dynamics of cellular pathways have a large role in function. Before recent advances, it has been very difficult to test the effects of these dynamics on cellular function; the control of local distribution of specific compounds to arbitrary positions within the cell has largely been inaccessible to researchers. To this effect, we have created a novel technique for the simple conjugation of bioactive elements to directable superparamagnetic nanoparticles. These nanoparticles are internalized in less than 3 hours and, through the use of a magnetizing ratcheting substrate, can be finely controlled within the cell. Thus with this technique it is possible to study single cells, proteins and pathways in a highly parallel manner. Using this technique, we are able to test the effect of varying concentrations of polylysine conjugated to nanoparticles. We have been able to show an overall increase in endosomal/lysosomal escape with increasing concentration of conjugated polylysine, suggesting that polylysine may play a role in the transfer of material from within vesicles to cellular plasma.

Abstract Title: Directed Evolution of a Binding Protein with High Affinity to R-Phycoerythrin Using Yeast Surface Display and Its Application in Live Cell Imaging

Abstract: Since the rational design and engineering of proteins are limited by our understanding of protein structure, function and dynamics, directed evolution utilizing iterative cycles of mutagenesis and selection has become a powerful tool for protein engineering. For mutagenesis, the libraries of millions of mutants are generated by various methods, including error-prone PCR, site-saturation mutagenesis and DNA shuffling. For selection, these mutants displayed at the outer surface of yeasts are screened by the fluorescence activated cell sorting (FACS) technology in a high throughput fashion. In this work, we have optimized the binding capability of the modified human fibronectin type III (monobody) toward R-Phycoerythrin, a red fluorescent protein, by using directed evolution. A large diversity of monobody variants was introduced by site-saturation mutagenesis with degenerate synthetic oligonucleotides on its two loops to generate the yeast display library. The selection of our desired protein was achieved by FACS. After several rounds of screening, a clone with high binding affinity to R-Phycoerythrin was obtained. With the significantly improvement of this binding protein, we can further apply it into live cell imaging.

Abstract Title: Distinct Roles of E1 Heterodimer (APPBP1 and UBA3) in NEDD8 Activation

Abstract: Ubiquitin and Ubiquitin like proteins (Ubls), such as NEDD8 and SUMO, play critical roles in various physiological and pathological processes, ranging from signal transduction, cell cycle to tumorgenesis. The Ubls are conjugated to their respective substrates by similar but distinct multi enzyme cascades that involve sequential actions of the E1 activating enzymes, E2 conjugating enzymes and E3 ligases. Although all the Ubls are conjugated to their substrates through a similar enzymatic cascade, the requirement for either single or heterodimer of E1 activating enzymes in various Ubl conjugation cascade is still a mystery. Here we report a significant discovery of the requirements of each subunit of E1 heterodimer for NEDD8 activation. We, for the first time, systematically dissect the NEDD8 activation step in real time by employing our new quantitative Forster Resonance Energy Transfer (FRET) technology. These studies show that APPBP1 (a subunit of NEDD8 E1 heterodimer) is not absolutely required for the NEDD8 activation. This discovery is significantly contrast with Ubiquitin and SUMO activations, which require single or heterodimers of E1 for activations. These findings provide not only detail molecular mechanisms of Ubl activation, but also potential evolution process at molecular level.

Abstract Title: Brain-Controlled Functional Electrical Stimulation in Stroke Survivors

Abstract: Despite the prevalence of stroke-induced gait impairment due to foot drop, current rehabilitative practices to improve gait function are limited, and orthoses can be uncomfortable and do not provide long-lasting benefits. Therefore, novel modalities that may facilitate lasting neurological and functional improvements, such as brain-computer interfaces (BCIs), have been explored. Here we assess the feasibility of BCI-controlled functional electrical stimulation (FES) as a novel physiotherapy for post-stroke foot drop. Three chronic stroke survivors with foot drop received three, 1-hour sessions of therapy during 1 week. All subjects were able to purposefully operate the BCI-FES system in real time. Furthermore, the salient electroencephalographic (EEG) features used for classification by the data-driven methodology were determined to be physiologically relevant. Over the course of this short therapy, the subjects’ dorsiflexion active range of motion (AROM) improved by 3, 4, and 8, respectively. These results indicate that chronic stroke survivors can operate the BCI-FES system, and that BCI-FES intervention may promote functional improvements.

Abstract Title: Studies of neuronal response to laser microbeam-induced site-specific axonal damage

Abstract: Studies of nerve regeneration and its biological basis are of key importance in development of novel therapeutic techniques for human neurodegenerative diseases and traumas. These studies require noninvasive in vitro methods to produce precise and controlled injury sites on axons and analyzing subsequent nerve regeneration responses. In this study, a 532nm Nd:YVO4 nanosecond laser was used to generate controlled site-specific damages on axons of primary hippocampal neuron cultures from rat embryos in vitro. The laser beam was coupled to an inverted microscope and focused by a high numerical aperture objective (NA=1.4) to a diffraction-limited spot of ˜0.46 µm diameter, below the 1-2 µm thickness of the axonal shaft. The neurons were cultured on glass bottom laminin-coated Petri dishes and grown 2-3 days in vitro prior to experiments. The localized laser microbeam sub-axotomy lesions were inducted on axons by application of laser macropulses of 600 12ns micropulses, with total energy of 360 µJ and peak power density of 14.6×109 W/cm2. The post-irradiation response of neurons was time-lapse recorded using a CCD camera under phase-contrast and bright-field illumination. The axonal shaft demonstrated immediate radiant energy-dependent thinning at the irradiation site followed by restoration to pre-irradiation thickness after 2-3 minutes. The preliminary results demonstrated formation of transient and sustained preferential filopodial protrusions at or near the damage site. The growth cone of the axons responded by extending long filopodia in a retrograde direction towards the lesion site. The axons also responded by ceasing forward progression and turning towards the injury site. The observations suggest existence of guidance cues released from the damage site detected by the sensory growth cone filopodia and subsequent enhanced actin-polymerization at the leading edge of the growth cone. This leads to outgrowth and turning towards the damage site.

Abstract Title: The Development of SHP2 Activator for Live Cells

Abstract: Src Homology 2 (SH2) Domain-containing Protein Tyrosine Phosphatase 2 (SHP2) is a ubiquitous tyrosine kinase playing crucial roles in various cellular processes. Its mutation can cause different diseases, such as Noonan syndrome and leukemia. It has been revealed that the enzymatic activity of SHP2 is the central target of abnormal regulation during these disease developments. While SHP2 has been well studied on its activation mechanism, there is a lack of method to directly activate it in live cells to provide a tool for the investigation of SHP2 activity in regulating cellular functions. In this work, we generated a SHP2 activator based on a hetero-dimerization system, which enables the elevation of SHP2 activity with a fast fashion in live cells.

Abstract Title: The structural basis of DNA repair regulation by Ku70-Ku80 acetylation

Abstract: Of the various forms of DNA damage repair mechanisms, non-homologous end-joining (NHEJ) is the most susceptible mechanism to result in chromosomal translocation and abnormalities. Tremendous amount of work has been done on the spatiotemporal orchestration and formation of DNA damage assembly, in particular on the role of post-translational modifications in recruitment of the DNA damage repair factors. However, very little is understood about the structural mechanism of such modifications on the acquisition of chromosomal abnormalities during NHEJ. Post-translational modifications (PTMs) have been widely studied in the context of NHEJ and are deemed critical for proper DNA damage repair. We focus on the role of acetylation on the functional mechanism of the first recruited protein complex to the site of damage, Ku70-Ku80. Through molecular dynamics (MD) simulations and free energy (FE) calculations, we determined the effect of lysine acetylation on Ku70-Ku80 dynamics and conformational transitions. The acetylation of Ku70 is believed to regulate its function and affect the DNA repair process. Nine lysine residues have been identified as targets for acetylation in vivo. Five of these residues lie in a flexible C-terminal linker of Ku70, while the remaining four are in the DNA binding region of the core domain. Electrostatic potential calculations reveal the presence of alternating positively and negatively charged patches in the C-terminal linker and two oppositely charged surfaces in the C-terminal SAP domain of Ku70. MD simulations show formation and deformation of intra- and inter-molecular salt-bridges and charge driven conformational transitions, illustrating coupling of dynamics and electrostatics. We will discuss charge removal by acetylation of lysine residues in view of conformational changes and function.

Abstract Title: A single-chip silicon array of 65k integrate-and-fire neurons with dynamically reconfigurable synaptic connectivity

Abstract: We present an 65k-neuron integrate-and-fire array transceiver (IFAT) for spike-based neural computation with low-power, high-throughput reconfigurable connectivity. The internally analog, externally digital chip is fabricated on a 4×4 die in 90nm CMOS and arranged in 4 quadrants of 16k parallel addressable neurons. Each neuron circuit serves input spike events by dynamically instantiating conductance-based synapses onto four local synapse circuits over two membrane compartments, and produces output spike events upon reaching a threshold in integration over one of the membrane compartments. Fully asynchronous input and output spike event data streams are mediated over the standard address event representation (AER) protocol. To support full event throughput at large synaptic fan-in, a two-tier micro-pipelining scheme parallelizes input events along neural array cores, and along rows of each core. Measured results show sustained peak synaptic event throughput of 17.2 Mevents/s per quadrant, at 19.2 pJ average energy per synaptic input event and 25 uW standby power.

Abstract Title: Nanochannel Trap Arrays for Monitoring Single Mitochondrion Behavior

Abstract: Using nanofluidic channels in PDMS of cross section 500 nm x 2 µm, we demonstrate the trapping and interrogation of individual, isolated mitochondria. Fluorescence labeling demonstrates the immobilization of mitochondria at discrete locations along the channel. Interrogation of mitochondrial membrane potential with different potential sensitive dyes (JC-1 and TMRM) indicates the trapped mitochondria are vital in the respiration buffer. Fluctuations of the membrane potential can be observed at the single mitochondrial level. A variety of chemical challenges can be delivered to each individual mitochondrion in the nanofluidic system. As sample demonstrations, increases in the membrane potential are seen upon introduction of OXPHOS substrates into the nanofluidic channel. Introduction of Ca2+ into the nanochannels induces mitochondrial membrane permeabilization (MMP), leading to depolarization, observed at the single mitochondrial level. A variety of applications in cancer biology, stem cell biology, apoptosis studies and high throughput functional metabolomics studies can be envisioned using this technology(1).

Abstract Title: A Supervised Multi-Sensor Matched Filter for the Detection of Extracellular Action Potentials

Abstract: Multi-sensor extracellular recording entails using several proximally spaced electrode channels to record from multiple neurons at the same time. Although this approach increases the breadth of recorded electrophysiological data, the resulting low signal-to-noise ratio (SNR) makes signal detection very difficult. Matched filters are a popular and effective detection technique for neurophysiological data, however, there aren’t many applications of this technique adapted for multi-sensor data. A matched filter was therefore designed to better detect extracellular action potentials (EAPs) from multi-sensor extracellular recordings. The detector was tested on tetrode data from a locust antennal lobe and assessed against three trained analysts. Half of the data was used as training data, and the remaining half as test data. 25 EAPs and noise samples were selected manually from the training data and used to train the filter. To reduce complexity, the filter assumed that the underlying noise in the data was spatially white. The detector performed with average true positive (TP) and false positive (FP) rates of 84.62% and 16.63% respectively. Furthermore, most of the spikes identified as FPs when compared against analysts 1 and 2, were identified as TPs by analyst 3. This indicates that the FP rate is largely driven by the relatively arbitrary and inconsistent thresholds set by a given analyst. Overall, the detector’s high level of performance indicates the algorithm is suitable for widespread use.

Abstract Title: Network Analysis of Intra-Molecular Interactions of the HIV-1 gp120 V3 Loop

Abstract: The V3 loop is a contact point with which HIV attaches to its target cells by interacting with co-receptors CCR5 or CXCR4 depending on the stages of infection or disease. HIV, along with the V3 loop, is constantly mutating in sequence and is highly flexible in structure, but it continues to recognize and show preference towards these receptors. It has been previously proposed that the mechanism of recognition involves charge complementarity between the V3 loop and the extracellular domain of CCR5/CXCR4. The objective of this study is to utilize network theory to analyze networks of intra-molecular interaction within the V3 loop to search for and correlate persistency and differences in network properties to how they affect the V3 loop stability and co-receptor selectivity. Contact maps and hydrogen bonds from molecular dynamics simulations of two V3 loops with similar sequences, but different structures were used to define network nodes and edges for our analysis. Community analysis was used to identify intra-molecular communication within and between the different sections of the V3 loop, the base, the stem, and the tip. The network analysis confirms previous studies and provides new insights on the role of critical amino acids for the stability and co-receptor interaction as well as possible allosteric signaling. This work may be useful for the mechanistic understanding of viral entry at molecular level as well as for inhibitor design of HIV-1 entry into cells and contribute towards potential treatment for HIV-1.

Abstract Title: MICROTUBULE-ASSOCIATED PROTEIN EB1 BINDS COOPERATIVELY, DIFFUSES ALONG THE MICROTUBULE LATTICE, AND SLOWS KINESIN TRANSLOCATION IN VITRO

Abstract: We investigate the mechanisms and effects of EB1 binding on microtubules formed with the slowly hydrolyzable GTP analogs GMPCPP and GTP?S, which recapitulate the end binding behavior of EB1 along their entire length. We observe binding of an EB1-GFP construct to microtubules using TIRF microscopy and find that EB1 binds cooperatively, and in a manner that depends upon nucleotide analog and the presence of taxol. We can also directly observe the motions of single molecules of EB1-GFP. We find that individual EB1 proteins diffuse along the microtubule lattice, and that the diffusion coefficient depends on salt concentration and GTP analog. To explore whether EB1 presence and diffusion has an effect on kinesin-driven cargo transport, we observe quantum dot labeled kinesins walking on microtubules assembled with GMPCPP and taxol and coated with EB1. We find that the addition of EB1 significantly reduces kinesin speed compared to the no EB1 condition, and that this effect is concentration dependent. Our data suggest a new possible mechanism for the regulation of kinesin function by EB1 in which kinesin speed is directly modulated through the interference of EB1 diffusion.
Abstract Title: Noninvasive Brain-Computer Interface Driven Functional Electrical Stimulation System for Overground Walking

Abstract: Decreased physical activity and wheelchair reliance after spinal cord injury (SCI) typically cause a number of medical comorbidities. Brain-computer interfaces (BCI) may offer a novel solution to ambulation after SCI by allowing direct brain control of an external device, such as functional electrical stimulation (FES) systems. To this end, we developed a noninvasive electroencephalogram (EEG)-based BCI-controlled FES system (Parastep I System, Sigmedics, Fairborn, OH) for overground walking. We tested the performance of this system in a SCI participant with paraplegia.

To assess feasibility, EEG was recorded for 10 min while the SCI participant performed alternating epochs of attempted walking and idling to develop a prediction model that classifies EEG in real time. An FES system for overground walking (Parastep I) was then integrated with the BCI to enable wireless BCI driven walking and standing while being weight loaded (ZeroG, Aretech LLC., Ahsburn, VA). The participant performed several goal-oriented walking tasks where he used the BCI-Parastep system to walk 12 ft (3.6 m) while making 3 stops for 10-20 s at cones positioned 6 ft apart. Physical sensors (gyroscopes, laser) were used to assess the performance of the subject in this task via cross-correlation and information transfer rate (ITR) analyses. The SCI participant with paraplegia (male, age 26, T6, ASIA B) was able to purposefully operate the BCI-Parastep system in real time. After 12 sessions over 7 days, he was able to perform the task with a 0.66±0.19 correlation at a 4.34±4.63 s lag, and a 1.68±0.94 bits/s ITR. He also performed the task with no omissions and very few false alarms (<0.001 FAs/min).This study demonstrates the first BCI system for overground ambulation after SCI. If successful in a larger population of SCI individuals, this system may become the first restorative treatment for overground walking after paraplegia due to SCI.

Abstract Title: Directed Evolution of Anti-HIV Antibodies Using an Expanded Genetic Code

Abstract: Sulfated tyrosine residues play a critical role in a large set of human antibodies against HIV that function by binding gp120. The sulfates are installed posttranslationally. Therefore, the sequences surrounding target tyrosines are restricted to specific motifs that recruit sulfation machinery, but these surrounding sequences may be suboptimal for gp120 binding. We hypothesize that removing these sequence constraints may allow for better sulfated HIV antibodies with enhanced binding affinity for gp120. We are able to test this by incorporating sulfotyrosines cotranslationally as genetically encoded unnatural amino acids (UAA’s). Here we show the use of an orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair optimized for sulfotyrosine incorporation in a phage display antibody evolution platform. Up to five sulfotyrosines were successfully incorporated in phage displaying E51, a known sulfated antibody, with minimal reduction in phage expression. We plan to create E51 antibody libraries randomized in sequences surrounding sulfotyrosines to select for better antibodies

Abstract Title: Pairwise reconstruction of hippocampal subregions to decode learning and memory: EC-DG, DG-CA3, CA3-CA1

Abstract: Sub-regions of the hippocampus are thought to contribute differently to stages of learning and memory, but we don’t yet know the code for how this information is created. Our goal was to determine if these regions could be distinguished as they self-wire into the anatomically accurate tri-synaptic network from the entorhinal cortex (EC) to the dentate gyrus (DG) to the CA3 or to the CA1, in pairs. We reconstructed paired components of the tri-synaptic pathway: EC-DG, DG-CA3, CA3-CA1 in culture by microdissection of rat hippocampus. We confined each subregion in a compartment connected by 51 microtunnels each 10 µm wide. This device was aligned with a 60 electrode array (MEA). Neurons from subregions of the rat hippocampus were dissected and plated into separate subcompartments. Spiking activity was recorded after network development of 3 weeks in culture and analyzed for differences in activity dynamics.  Compared to controls of DG on each side, a DG subregion connected to EC showed lower spike rates and burst rates. With EC connected to DG, the EC subregion showed even lower spike and burst rates but now spikes per burst and extraburst spike rates were also lower. These differences occurred with equal percent active electrodes and burst durations. For the DG-CA3 pair, CA3 was generally less active than DG, except for a higher rate of intraburst spikes in the CA3 than the DG. For the CA3-CA1 pair, the lack of input from DG yielded lower CA3 spike and burst rates, while CA1 rates were higher than CA3. Extension to a closed-loop 4 compartment model will be described. This technology will enable determination of the network integration of stimulation-dependent plasticity and how subregion-specific information patterns are reliably transmitted but differentially processed within each hippocampal subregion. (Supported by the NIH under Grant NS052233)

Abstract Title: Engineering cellulose-degrading complexes from anaerobic gut fungi

Abstract: Anaerobic gut fungi are primary colonizers and decomposers of plant biomass in monogastric herbivores and ruminants. Their ability to hydrolyze complex lignocellulosic materials without pretreatment is of great value to cellulosic bioenergy development, where breakdown of plant biomass is a stringent bottleneck. It is believed that large cellulolytic complexes, or cellulosomes, mediate this ability. While bacterial cellulosomes are well characterized, the molecular architecture of fungal cellulosomes is poorly understood. Here, we characterized the composition and structure of fungal cellulosomes from a representative group of gut fungi. To do so, we developed a rapid approach to isolate cellulosomes from fungal cultures, and following the purification of these cellulosomes, we combined mass spectrometry with strand-specific transcriptomic sequencing to identify the key components of these cellulosomes. To further investigate complex formation among these proteins, we used size-exclusion chromatography to show that secreted cellulases assemble in large complexes. We also provide evidence that non-catalytic dockerin domains (NCDDs) encoded within cellulases mediate enzyme self-assembly. Furthermore, we show that NCDDs have inter- and intra-species promiscuity, interacting with multiple cellulases from the same and other species of gut fungi. This suggests a departure from the paradigm of bacterial cellulosomes that is centered on a non-catalytic scaffoldin. A better understanding of plant biomass breakdown in gut fungi has the potential to transform consolidated bioprocessing platforms that aim to convert plant biomass to energy.

Abstract Title: Event-Driven Contrastive Divergence for Spiking Neuromorphic Systems

Abstract: Restricted Boltzmann Machines (RBMs) and Deep Belief Networks have been demonstrated to perform efficiently in a variety of applications, such as dimensionality reduction, feature learning, and classification. Their implementation on neuromorphic hardware platforms emulating large-scale networks of spiking neurons can have significant advantages from the perspectives of scalability, power dissipation and real-time interfacing with the environment. However the traditional RBM architecture and the commonly used training algorithm known as Contrastive Divergence (CD) are based on discrete updates and exact arithmetics which do not directly map onto a dynamical neural substrate. Here, we present an event-driven variation of CD to train a RBM constructed with Integrate & Fire (I&F) neurons, that is constrained by the limitations of existing and near future neuromorphic hardware platforms. Our strategy is based on neural sampling, which allows us to synthesize a spiking neural network that samples from a target Boltzmann distribution. The recurrent activity of the network replaces the discrete steps of the CD algorithm, while Spike Time Dependent Plasticity (STDP) carries out the weight updates in an online, asynchronous fashion.
We demonstrate our approach by training an RBM composed of leaky I&F neurons with STDP synapses to learn a generative model of the MNIST hand-written digit dataset, and by testing it in recognition, generation and cue integration tasks. Our results contribute to a machine learning-driven approach for synthesizing networks of spiking neurons capable of carrying out practical, high-level functionality.

Abstract Title: Investigating the Binding Mode of C3d–CR2 Using Poisson-Boltzmann Electrostatic Calculations and Molecular Dynamics Simulations

Abstract: The complement system, consisting of several plasma proteins, is an important part of the innate immune system. The interaction of complement fragment C3d and complement receptor 2 (CR2) plays a crucial role as a link between innate and adaptive immunity, leading to enhancement of B cell mediated antibody production during initial complement response to infection. Over the past decade, there has been much debate over the mode of binding between C3d and CR2 [1]. The first binding mode (PDB code: 3OED) has an acidic patch at the binding site whereas the second binding site (PDB code: 1GHQ) is further away and lacks such an acidic patch. However, it is believed that the second binding site may not have physiological significance and that it is influenced by crystallization conditions. We used an array of computational tools to gain insight into the binding mode between C3d and CR2. In order to elucidate the effect of electrostatics on the two binding sites, we performed computational alanine scans and used Poisson-Boltzmann electrostatic calculations to predict the electrostatic binding free energy of protein mutants. We have also performed individual computational mutagenesis of previous physiologically generated mutants from the literature, to evaluate the effect of these mutations on binding of each complex in light of experimental data. In addition, we used molecular dynamics (MD) simulations to gain further insight into the binding nature of C3d – CR2. Our electrostatic similarity analysis and MD simulations indicate that the binding site at the acidic patch is more stable, with more favorable interactions at that interface. Furthermore, computational mutagenesis data for this binding mode better correlates with corresponding experimental data. With these results, we hope to bring closure to this controversy.
[1] Van den Elsen, JMH, Isenman DE (2011) Science 332.6029: 608–611.

Abstract Title: Strategies for Improving Inner Neuron Survivability Post Implantation of A PDMS Based Retinal Prosthesis Interface

Abstract: Normal vision and retinal function is lost due to the degeneration of the photoreceptor cell layer caused by diseases such as Age-Related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP). It is possible to restore functional vision through the implantation of a retinal prosthesis device into the subretinal space. A class of these devices use polymers such as polydimethylsiloxane, paralyene, polyimide or toher. The substrate layer acts to give both structural integrity as well as electrical insulation the neural stimulation electrodes. Unfortunately, implantation of a prosthesis into the subretinal space results the blockage the transepithelial nutrient flow from the retinal pigment epithelium (RPE) to the sub-choroid space. This disruption results in degradation of inner neurons and fibrosis.   Modifying the PDMS layer to be more permeable to the required nutrients can alleviate this nutrient blockage. This study aims to increase the porosity of the PDMS layer using novel preparation techniques for the plastic layer. The first preparation consists of an emulsion technique that creates micro-bubbles. The second preparation thins the PDMS layer by varying the ratio of elastomer base to curing agent. These preparations have been shown to improve the diffusion of proteins and essential nutrients through the layer. In vitro neural functionality studies have shown that this preparation method increases the longevity of retinal cell layers. Further studies are proposed to investigate the interaction of these preparation methods and the effect they may have on mechanical and electrical properties of the plastic substrate. This study demonstrates the viability of this method to improve neural cell survivability for PDMS based neural interfaces.

Abstract Title: Functional and histological evaluation of a high-density optoelectronic nanowires in rabbits.

Abstract: Several engineering solution have been developed to replace the function of degenerated photoreceptors in the neural retina due to age related macular degeneration (AMD) and retinitis pigmentosa (RP). The most successful systems rely on micro­photodiode for light detection or cameras to capture the image with downstream processing and power before stimulating retinal neurons. The use of photodetectors to detect light inside the eye, allows the prosthetic device to make use of the eye’s natural object tracking and reduce some of the signal processing requirements. We have developed high quantum efficiency photovoltaic silicon nanowire photodetectors that can detect light and stimulate neurons with high density and a large area of stimulation. In this work we evaluated the feasibility of subretinal implantation and neural retinal electrical stimulation with these devices in rabbits by measuring evoked responses in visual cortex

Abstract Title: Vascular Stents with Rationally-Designed Sub-Micrometer Scale Surface Patterning

Abstract: Drug-eluting stents have revolutionized the field of interventional cardiology by reducing incidence of restenosis through local delivery of drugs that inhibit inflammation caused by implantation-induced injury. However, growing evidence suggests that this may also inhibit reestablishment of the endothelium, thus delaying healing and increasing potential for thrombogenic stimulus. Herein, we discuss our recent progress towards realization of next-generation titanium (Ti) stents that seek to mitigate adverse physiological responses to stenting via rational design of stent surface topography at the micro- and nanoscale.

Specifically, we discuss: 1) advances which now allow patterning of features in Ti substrates down to 150 nm, which represents the smallest features achieved to date using our novel Ti deep reactive ion etching (Ti DRIE) technique; 2) creation and evaluation of balloon-deployable, cylindrical, surface-patterned stents from micromachined planar Ti substrates; and 3) integration of these processes to produce a device platform that allows, for the first time, evaluation of surface patterning in more physiologically relevant contexts, e.g. in vitro organ culture. Collectively, these efforts represent key steps towards our long-term goal of developing a new paradigm for stents in which rationally-designed surface patterning provides a physical means for complementing, or replacing, current pharmacological interventions.

Abstract Title: Spatio-temporal Mapping of Mercury Contamination using a Smartphone

Abstract: We demonstrate a cost-effective handheld mercury detection platform installed on a smartphone for spatio-temporal mapping of mercury contamination in water samples. This platform integrates an opto-mechanical attachment (< 40 grams) to the camera module of a smartphone to digitally quantify colorimetric transmission signals of a mercury-specific gold nanoparticle (Au NP) assay running on the phone [1]. This Au NP assay displays a distinct color change from red to blue in the presence of mercury ions due to the binding ‘competition’ of a mercury-specific aptamer sequence between mercury ions and positively charged Au NPs. The transmission intensities of this assay are quantified ratiometrically by illuminating the sample tubes with two LEDs at red (523 nm) and green (625 nm) wavelengths simultaneously. A custom-developed Android application was also installed on the smartphone for rapidly processing the acquired images and displaying the test results within less than 7 seconds. The limit of detection of mercury ions was determined to be ~3.5 parts-per-billion (ppb), which is on the same order of magnitude with the US EPA and WHO’s mercury reference concentration in drinking water (2 ppb and 6 ppb, respectively). Spatio-temporal mapping of mercury contamination at over 50 locations in California was also demonstrated.

[1] Q. Wei, R. Nagi, K. Sadeghi, S. Feng, E. Yan, S. J. Ki, R. Caire, D. Tseng, and A. Ozcan. ACS Nano, 2014, 8, 1121-1129.

Abstract Title: Binding Kinetic Rates Measured via Electrophoretic Band Crossing in a Pseudohomogeneous Format:

Abstract: With relevance spanning from disease diagnostics such as immunohistochemistry to immunoassays and therapeutics, antibody reagents play critical roles in the life sciences, clinical chemistry, and clinical medicine. Nevertheless, non-specific interactions, low sensitivity, and performance reproducibility remain problematic. Selecting antibodies based on their antigen binding kinetic properties, such as their association and dissociation rate constants, kon and koff, can provide a quantitative metric that can further optimize and validate immunoreagent selection. These rate constants quantify the ability for an antibody to associate (bind) or dissociate (unbind) to a target analyte and determines inherent binding strength. Therefore a metric such as this has the power to eliminate problems seen by clinicians, researchers and drug developers alike in regards to false positive, false negatives, and problems with reproducibility seen in antibody-based approaches and inform in assay design. Consequently, scalable and efficient analytical tools for informed selection of reliable antibody reagents would have wide impact. Here we introduce a Kinetic Polyacrylamide Gel Electrophoresis (KPAGE) microfluidic assay that directly measures antibody-antigen association and dissociation rate constants, kon and koff. To study antibody-antigen association, an antigen zone is electrophoresed through a zone of immobilized antibody. Upon crossing, the interaction yields a zone of immobilized immunocomplex, To quantify kon, we assess immunocomplex formation for a range of antigen-antibody interaction times. Here, interaction time is controlled by the velocity of the electromigrating antigen zone, which is determined by the strength of the applied electric field. All species are fluorescently labeled. To quantify koff, an immobilized zone of immunocomplex is subjected to in situ buffer dilution, while measuring the decay in immunocomplex concentration.