Scholarly Works, School of Biomedical Engineering and Sciences

Permanent URI for this collection

https://hdl.handle.net/10919/23275

Research articles, presentations, and other scholarship

Browse

Now showing 1 - 14 of 14

Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers
Najem, Joseph S.; Dunlap, Myles D.; Rowe, Ian D.; Freeman, Eric C.; Grant, John Wallace; Sukharev, Sergei; Leo, Donald J. (Springer Nature, 2015-09-08)
MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.
Biomechanics of hair cell kinocilia: experimental measurement of kinocilium shaft stiffness and base rotational stiffness with Euler-Bernoulli and Timoshenko beam analysis
Spoon, Corrie E.; Grant, John Wallace (Company of Biologists, 2011-03-01)
Vestibular hair cell bundles in the inner ear contain a single kinocilium composed of a 9+2 microtubule structure. Kinocilia play a crucial role in transmitting movement of the overlying mass, otoconial membrane or cupula to the mechanotransducing portion of the hair cell bundle. Little is known regarding the mechanical deformation properties of the kinocilium. Using a force-deflection technique, we measured two important mechanical properties of kinocilia in the utricle of a turtle, Trachemys (Pseudemys) scripta elegans. First, we measured the stiffness of kinocilia with different heights. These kinocilia were assumed to be homogenous cylindrical rods and were modeled as both isotropic Euler-Bernoulli beams and transversely isotropic Timoshenko beams. Two mechanical properties of the kinocilia were derived from the beam analysis: flexural rigidity (El) and shear rigidity (kGA). The Timoshenko model produced a better fit to the experimental data, predicting El=10,400 pN mu m(2) and kGA=247 pN. Assuming a homogenous rod, the shear modulus (G=1.9 kPa) was four orders of magnitude less than Young's modulus (E=14.1 MPa), indicating that significant shear deformation occurs within deflected kinocilia. When analyzed as an Euler-Bernoulli beam, which neglects translational shear, El increased linearly with kinocilium height, giving underestimates of El for shorter kinocilia. Second, we measured the rotational stiffness of the kinocilium insertion (kappa) into the hair cell's apical surface. Following BAPTA treatment to break the kinocilial links, the kinocilia remained upright, and kappa was measured as 177 +/- 47 pN mu m rad(-1). The mechanical parameters we quantified are important for understanding how forces arising from head movement are transduced and encoded by hair cells.
Cytoskeletal Disruption after Electroporation and Its Significance to Pulsed Electric Field Therapies
Graybill, Philip M.; Davalos, Rafael V. (MDPI, 2020-04-30)
Pulsed electric fields (PEFs) have become clinically important through the success of Irreversible Electroporation (IRE), Electrochemotherapy (ECT), and nanosecond PEFs (nsPEFs) for the treatment of tumors. PEFs increase the permeability of cell membranes, a phenomenon known as electroporation. In addition to well-known membrane effects, PEFs can cause profound cytoskeletal disruption. In this review, we summarize the current understanding of cytoskeletal disruption after PEFs. Compiling available studies, we describe PEF-induced cytoskeletal disruption and possible mechanisms of disruption. Additionally, we consider how cytoskeletal alterations contribute to cell–cell and cell–substrate disruption. We conclude with a discussion of cytoskeletal disruption-induced anti-vascular effects of PEFs and consider how a better understanding of cytoskeletal disruption after PEFs may lead to more effective therapies.
Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis
Salmanzadeh, Alireza; Kittur, Harsha; Sano, Michael B.; Roberts, Paul C.; Schmelz, Eva M.; Davalos, Rafael V. (American Institute of Physics, 2012-06-01)
Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3699973] Actual pdf downloaded from NCBI.
Distinguishing between overdrive excited and suppressed ventricular beats in guinea pig ventricular myocardium
Greer-Short, Amara D.; Poelzing, Steven (Frontiers, 2015-02-18)
Rapid ventricular pacing rates induces two types of beats following pacing cessation: recovery cycle length (RCL) prolongation (overdrive suppression) and RCL shortening (overdrive excitation). The goals of this study were to compare common experimental protocols for studying triggered activity in whole-heart preparations and differentiate between recovery beats using a new methodology. Post-pacing recovery beat cycle length (RCL) and QRS were normalized to pre-paced R-R and QRS intervals and analyzed using a K-means clustering algorithm. Control hearts only produced suppressed beats: RCL ratio increased with rapid pacing (25 +/- 4.0%, n = 10) without changing QRS duration. Rapid pacing during hypercalcemia + hypothermia (5.5 mM and 34 degrees C) produced significantly earlier excited beats (53 +/- 14%, n = 5) with wider QRS durations (58 +/- 6.3%, n = 5) than suppressed beats. Digoxin + hypothermia (0.75 mu M) produced the most excited beats with significantly earlier RCL (44 +/- 3.2%, n = 6) and wider QRS (60 +/- 3.1%, n = 6) ratios relative to suppressed beats. Increasing pacing further shortened RCL (30 +/- 7.8%, n = 6). In a prospective study, TTX (100 nM) increased RCL ratio (15 +/- 6.0%, n = 10) without changing the QRS duration of excited beats. The algorithm was compared to a cross-correlation analysis with 93% sensitivity and 94% specificity. This ECG based algorithm distinguishes between triggered and automatic activity.
Endothelial cell sensing, restructuring, and invasion in collagen hydrogel structures
Hosseini, Yahya; Agah, Masoud; Verbridge, Scott S. (The Royal Society of Chemistry, 2015-09-03)
Experimental tools to model cell-tissue interactions will likely lead to new ways to both understand and treat cancer. While the mechanical properties and regulation of invasion have been recently studied for tumor cells, they have received less attention in the context of tumor vascular dynamics. In this article, we have investigated the interaction between the surfaces of structures encountered by endothelial cells invading their surrounding extracellular matrix (ECM) during angiogenesis. For this purpose, we have fabricated round and sharp geometries with various curvature and sharpness indices in collagen hydrogel over a wide range of stiffness to mimic different microenvironments varying from normal to tumor tissues. We have then cultured endothelial cells on these structures to investigate the bi-directional interaction between the cells and ECM. We have observed that cell invasion frequency is higher from the structures with the highest sharpness and curvature index, while interestingly the dependence of invasion on the local micro-geometry is strongest for the highest density matrices. Notably, structures with the highest invasion length are linked with higher deformation of side structures, which may be related to traction force-activated signaling suggesting further investigation. We have noted that round structures are more favorable for cell adhesion and in some cases round structures drive cell invasion faster than sharp ones. These results highlight the ability of endothelial cells to sense small variations in ECM geometry, and respond with a balance of matrix invasion as well as deformation, with potential implications for feedback mechanisms that may enhance vascular abnormality in response to tumor-induced ECM alterations.
Folic Acid-Conjugated Cellulose Nanocrystals Show High Folate-Receptor Binding Affinity and Uptake by KB and Breast Cancer Cells
Bittleman, Katelyn Rose; Dong, Shuping; Roman, Maren; Lee, Yong Woo (American Chemical Society, 2018-10-24)
The study evaluates cellulose nanocrystals (CNCs) as nanocarriers for targeted, intracellular delivery of molecular agents. CNCs were labeled with fluorescein-5′-isothiocyanate as an imaging agent and conjugated to folic acid (FA) as a targeting ligand. The CNC conjugates were characterized by UV–vis spectroscopy, ζ-potential analysis, dynamic light scattering, and atomic force microscopy. Cellular binding/uptake of the FA-conjugated CNCs by KB and MDA-MB-468 cells was quantified with cellular uptake assays. Internalization of the particles was confirmed by confocal microscopy. Uptake mechanisms were determined by inhibition studies with chlorpromazine and genistein. Binding affinity was qualitatively assessed with a free folate inhibition assay. Both KB and MDA-MB-468 cells exhibited significant and folate-receptor specific binding/uptake of FA-conjugated CNCs. Clathrin-mediated endocytosis was a significant uptake mechanism in both cell types, whereas caveolae-mediated endocytosis only played a significant role in MDA-MB-468 cells. Uptake inhibition of FA-conjugated CNCs by KB cells required high concentrations (>1 mM) of free FA. The observed FR-specific internalization of FA-conjugated CNCs by FR-positive cancer cells and tumors and their remarkable high affinity for the FR demonstrate the great potential of CNCs as novel nanocarriers for imaging agents and chemotherapeutics in the early detection and treatment of cancer.
Investigating dielectric properties of different stages of syngeneic murine ovarian cancer cells
Salmanzadeh, Alireza; Sano, Michael B.; Gallo-Villanueva, R. C.; Roberts, Paul C.; Schmelz, Eva M.; Davalos, Rafael V. (American Institute of Physics, 2013-01-01)
In this study, the electrical properties of four different stages of mouse ovarian surface epithelial (MOSE) cells were investigated using contactless dielectrophoresis (cDEP). This study expands the work from our previous report describing for the first time the crossover frequency and cell specific membrane capacitance of different stages of cancer cells that are derived from the same cell line. The specific membrane capacitance increased as the stage of malignancy advanced from 15.39 +/- 1.54 mF m(-2) for a non-malignant benign stage to 26.42 +/- 1.22 mF m(-2) for the most aggressive stage. These differences could be the result of morphological variations due to changes in the cytoskeleton structure, specifically the decrease of the level of actin filaments in the cytoskeleton structure of the transformed MOSE cells. Studying the electrical properties of MOSE cells provides important information as a first step to develop cancer-treatment techniques which could partially reverse the cytoskeleton disorganization of malignant cells to a morphology more similar to that of benign cells. (C) 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4788921] Actual pdf downloaded from NCBI.
Irreversible electroporation using tissue vasculature to treat aberrant cell masses or create tissue scaffolds
(United States Patent and Trademark Office, 2018-01-16)
The present invention relates to the field of medical treatment of diseases and disorders, as well as the field of biomedical engineering. Embodiments of the invention relate to the delivery of Irreversible Electroporation (IRE) through the vasculature of organs to treat tumors embedded deep within the tissue or organ, or to decellularize organs to produce a scaffold from existing animal tissue with the existing vasculature intact. In particular, methods of administering non-thermal irreversible electroporation (IRE) in vivo are provided for the treatment of tumors located in vascularized tissues and organs. Embodiments of the invention further provide scaffolds and tissues from natural sources created using IRE ex vivo to remove cellular debris, maximize recellularization potential, and minimize foreign body immune response. The engineered tissues can be used in methods of treating subjects, such as those in need of tissue replacement or augmentation.
N-(3-oxododecanoyl)-L-homoserine lactone interactions in the breast tumor microenvironment: Implications for breast cancer viability and proliferation in vitro
Balhouse, Brittany N.; Patterson, Logan; Schmelz, Eva M.; Slade, Daniel J.; Verbridge, Scott S. (PLOS, 2017-07-10)
It is well documented that the tumor microenvironment profoundly impacts the etiology and progression of breast cancer, yet the contribution of the resident microbiome within breast tissue remains poorly understood. Tumor microenvironmental conditions, such as hypoxia and dense tumor stroma, predispose progressive phenotypes and therapy resistance, however the role of bacteria in this interplay remains uncharacterized. We hypothesized that the effect of individual bacterial secreted molecules on breast cancer viability and proliferation would be modulated by these tumor-relevant stressors differentially for cells at varying stages of progression. To test this, we incubated human breast adenocarcinoma cells (MDA-MB-231, MCF-DCIS.com) and non-malignant breast epithelial cells (MCF-10A) with N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule from Pseudomonas aeruginosa that regulates bacterial stress responses. This molecule was selected because Pseudomonas was recently characterized as a significant fraction of the breast tissue microbiome and OdDHL is documented to impact mammalian cell viability. After OdDHL treatment, we demonstrated the greatest decrease in viability with the more malignant MDA-MB-231 cells and an intermediate MCF-DCIS.com (ductal carcinoma in situ) response. The responses were also culture condition (i.e. microenvironment) dependent. These results contrast the MCF-10A response, which demonstrated no change in viability in any culture condition. We further determined that the observed trends in breast cancer viability were due to modulation of proliferation for both cell types, as well as the induction of necrosis for MDA-MB-231 cells in all conditions. Our results provide evidence that bacterial quorum-sensing molecules interact with the host tissue environment to modulate breast cancer viability and proliferation, and that the effect of OdDHL is dependent on both cell type as well as microenvironment. Understanding the interactions between bacterial signaling molecules and the host tissue environment will allow for future studies that determine the contribution of bacteria to the onset, progression, and therapy response of breast cancer.
A novel frequency analysis method for assessing K(ir)2.1 and Na (v)1.5 currents
Rigby, J. R.; Poelzing, Steven (2012-04)
Voltage clamping is an important tool for measuring individual currents from an electrically active cell. However, it is difficult to isolate individual currents without pharmacological or voltage inhibition. Herein, we present a technique that involves inserting a noise function into a standard voltage step protocol, which allows one to characterize the unique frequency response of an ion channel at different step potentials. Specifically, we compute the fast Fourier transform for a family of current traces at different step potentials for the inward rectifying potassium channel, K(ir)2.1, and the channel encoding the cardiac fast sodium current, Na(v)1.5. Each individual frequency magnitude, as a function of voltage step, is correlated to the peak current produced by each channel. The correlation coefficient vs. frequency relationship reveals that these two channels are associated with some unique frequencies with high absolute correlation. The individual IV relationship can then be recreated using only the unique frequencies with magnitudes of high absolute correlation. Thus, this study demonstrates that ion channels may exhibit unique frequency responses.
Performance enhancement of piezoelectric energy harvesters from wake galloping
Abdelkefi, Abdessattar; Scanlon, John Michael; McDowell, E.; Hajj, Muhammad R. (AIP Publishing, 2013-07-01)
Experiments are performed to investigate the effects of wake galloping on the range of flow speeds over which a galloping-based piezoaeroelastic energy harvester can be effectively used. Two different upstream cylinders and a wide range of spacing between the upstream and downstream cylinders are considered. Bifurcation diagrams and type of instability for different setups are determined. The results show a complex relation between the upstream circular cylinder size, the spacing between the two cylinders, the flow speed, and the load resistance on one hand, and the level of harvested power on the other hand. (C) 2013 AIP Publishing LLC.
Sex Differences in beta-Adrenergic Responsiveness of Action Potentials and Intracellular Calcium Handling in Isolated Rabbit Hearts
Hoeker, Gregory S.; Hood, A. R.; Katra, R. P.; Poelzing, Steven; Pogwizd, S. M. (PLOS, 2014-10-23)
Cardioprotection in females, as observed in the setting of heart failure, has been attributed to sex differences in intracellular calcium handling and its modulation by b-adrenergic signaling. However, further studies examining sex differences in badrenergic responsiveness have yielded inconsistent results and have mostly been limited to studies of contractility, ion channel function, or calcium handling alone. Given the close interaction of the action potential (AP) and intracellular calcium transient (CaT) through the process of excitation-contraction coupling, the need for studies exploring the relationship between agonist-induced AP and calcium handling changes in female and male hearts is evident. Thus, the aim of this study was to use optical mapping to examine sex differences in ventricular APs and CaTs measured simultaneously from Langendorff-perfused hearts isolated from naı¨ve adult rabbits during b-adrenergic stimulation. The non-selective b-agonist isoproterenol (Iso) decreased AP duration (APD90), CaT duration (CaD80), and the decay constant of the CaT (t) in a dosedependent manner (1–316.2 nM), with a plateau at doses $31.6 nM. The Iso-induced changes in APD90 and t (but not CaD80) were significantly smaller in female than male hearts. These sex differences were more significant at faster (5.5 Hz) than resting rates (3 Hz). Treatment with Iso led to the development of spontaneous calcium release (SCR) with a dose threshold of 31.6 nM. While SCR occurrence was similar in female (49%) and male (53%) hearts, the associated ectopic beats had a lower frequency of occurrence (16% versus 40%) and higher threshold (100 nM versus 31.6 nM) in female than male hearts (p,0.05). In conclusion, female hearts had a decreased capacity to respond to b-adrenergic stimulation, particularly under conditions of increased demand (i.e. faster pacing rates and ‘‘maximal’’ levels of Iso effects), however this reduced badrenergic responsiveness of female hearts was associated with reduced arrhythmic activity.
Temporal Characterization of Blood–Brain Barrier Disruption with High-Frequency Electroporation
Lorenzo, Melvin F.; Thomas, Sean C.; Kani, Yukitaka; Hinckley, Jonathan; Lee, Matthew; Adler, Joy; Verbridge, Scott S.; Hsu, Fang-Chi; Robertson, John L.; Davalos, Rafael V.; Rossmeisl, John H. Jr. (MDPI, 2019-11-23)
Treatment of intracranial disorders suffers from the inability to accumulate therapeutic drug concentrations due to protection from the blood–brain barrier (BBB). Electroporation-based therapies have demonstrated the capability of permeating the BBB, but knowledge of the longevity of BBB disruption (BBBD) is limited. In this study, we quantify the temporal, high-frequency electroporation (HFE)-mediated BBBD in an in vivo healthy rat brain model. 40 male Fisher rats underwent HFE treatment; two blunt tipped monopolar electrodes were advanced into the brain and 200 bursts of HFE were delivered at a voltage-to-distance ratio of 600 V/cm. BBBD was verified with contrast enhanced T1W MRI (gadopentetate dimeglumine) and pathologically (Evans blue dye) at time points of 1, 24, 48, 72, and 96 h after HFE. Contrast enhanced T1W scans demonstrated BBBD for 1 to 72 h after HFE but intact BBB at 96 h. Histologically, tissue damage was restricted to electrode insertion tracks. BBBD was induced with minimal muscle contractions and minimal cell death attributed to HFE. Numerical modeling indicated that brief BBBD was induced with low magnitude electric fields, and BBBD duration increased with field strength. These data suggest the spatiotemporal characteristics of HFE-mediated BBBD may be modulated with the locally applied electric field.

Browse

Browsing Scholarly Works, School of Biomedical Engineering and Sciences by Department "Biomedical Engineering and Mechanics"

Results Per Page

Sort Options