R-3 Repository :: Browsing by Author "Anvari, Bahman"

Browsing by Author "Anvari, Bahman"

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Analysis of mechanical characteristics of plasma membranes using optical tweezers
(2006) Ermilov, Sergey Alexandrovich; Anvari, Bahman
In this thesis work an optical tweezers system was designed and used to characterize viscoelastic response of plasma membranes (PMs) to an applied stress under different environmental conditions. In order to perform accurate force measurements we analyzed dynamics of an optically trapped microsphere under altering external viscous drag force using Fourier methods. Next, using optically trapped fluorescent microspheres, we recorded tethering force vs. PM displacement profiles, which revealed the tether formation process, initiated with linear deformation of the PM, followed by a nonlinear regime and terminated with the local separation of PM. Tethering force vs. displacement profiles were used to estimate tether formation force and stiffness parameter of the PM. Integration of the force-displacement profiles yielded the work of tether formation, including linear and nonlinear components. We characterized mechanical properties of the outer hair (OHC) and human embryonic kidney (HEK) cell PMs perfused with 10 mM sodium salicylate (Sal), which is known to affect electromotility of OHCs as well as PM surface charge, morphology of erythrocytes, and PM lipid diffusion. Sal reduced tether formation force, PM stiffness parameter, and equilibrium tethering force in HEK cells, explained by enhanced PM/cytoskeleton compliance. The parameters estimated for OHCs remained the same after Sal perfusion, which is consistent with the hypothesis that Sal induced reversible hearing loss appears to be more the result of its competition with essential anions and less the result of a change in PM mechanics. Finally, we found that cell perfusion with hypo- and hyperosmotic solutions did not influence equilibrium tethering force, attributed to cellular regulation of the effective PM tension. The observed reduction in magnitude of the fast tethering force relaxation process was related to the stress-minimizing redistribution of the PM lipids. Application of a non-specific transmembrane water transport blocker, mercury (II) chloride, increased steady-state and equilibrium tethering forces and suppressed slow component of the tethering force relaxation. Temporal tethering force profiles obtained from the same PM tethers pulled in several repetitive cycles exhibited different behavior and resulted in reduced forces and time constants explained by essential irreversibility of PM tether elongation involving PM-cytoskeleton dissociation and/or plastic deformations.
Charge-assembled capsules for phototherapy
(2012-12-25) Yu, Jie; Wong, Michael S.; Anvari, Bahman; Yaseen, Mohammad Abbas; Rice University; United States Patent and Trademark Office
Novel phototherapeutic methods and compositions are described herein. Nanoparticle-assembled microcapsules as a new type of delivery vehicle for photosensitive compounds may be synthesized through a two-step assembly process. Charged polymer chains and counterions may be combined with a photosensitive compound to form photosensitive aggregates, and then nanoparticles may be combined with the aggregates to form the microcapsules. The shell may be composed of nanoparticles and/or polymer, and the core interior may contain the photosensitive compound. Formation occurs rapidly (on the order of seconds) and the conditions are very mild (at room temperature, in aqueous solution, and at neutral pH). The microcapsule synthesis is highly suitable as an encapsulation method, particularly for a charged photosensitive molecule like ICG.
Measuring forces at the leading edge: a force assay for cell motility
(Royal Society of Chemistry, 2013) Farrell, Brenda; Qian, Feng; Kolomeisky, Anatoly; Anvari, Bahman; Brownell, William E.
Cancer cells become mobile by remodelling their cytoskeleton to form migratory structures. This transformation is dominated by actin assembly and disassembly (polymerisation and depolymerisation) in the cytoplasm. Synthesis of filamentous actin produces a force at the leading edge that pushes the plasma membrane forward. We describe an assay to measure the restoring force of the membrane in response to forces generated within the cytoplasm adjacent to the membrane. A laser trap is used to form a long membrane nanotube from a living cell and to measure the axial membrane force at the end of the tube. When the tube, resembling a filopodium, is formed and in a relaxed state the axial membrane force exhibits a positive stationary value. This value reflects the influence of the cytoskeleton that acts to pull the tube back to the cell. A dynamic sawtooth force that rides upon the stationary value is also observed. This force is sensitive to a toxin that affects actin assembly and disassembly, but not affected by agents that influence microtubules and myosin light chain kinase. We deduce from the magnitude and characteristics of dynamic force measurements that it originates from depolymerisation and polymerisation of F-actin. The on- and off-rates, the number of working filaments, and the force per filament (2.5 pN) are determined. We suggest the force-dependent transitions are thermodynamically uncoupled as both the on- and off-rates decrease exponentially with a compressive load. We propose kinetic schemes that require attachment of actin filaments to the membrane during depolymerisation. This demonstrates that actin kinetics can be monitored in a living cell by measuring force at the membrane, and used to probe the mobility of cells including cancer cells.
Modulation of mechanics and electromechanical force in prestin transfected membrane tethers
(2007) Zhang, Rui; Anvari, Bahman
The voltage-dependent movement, or electromotility, of cochlear outer hair cells (OHCs) contributes to cochlear amplification in mammalian hearing. Prestin, a transmembrane protein expressed in the lateral wall of the OHSs, is essential for electromotility, but molecular details of its function are unknown. Using a combined optical tweezers and whole-cell voltage clamping system, we have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) of membrane tethers, and related these parameters to prestin-associated non-linear capacitance (NLC). Our study included three test groups consisting of (i) untransfected; (ii) wild type (WT) prestin-transfected; and (iii) mutant (A100W) prestin-transfected human embryonic kidney (HEK) cells. While there was no difference in membrane effective viscosity among the three cell types, tethers from WT prestin-transfected HEK cells demonstrated altered membrane mechanical parameters and increased EMF values than control untransfected HEK cells. We found that A100W mutation in prestin, which eliminates NLC also diminishes EMF, but does not affect membrane mechanical parameters. These results suggest that prestin-associated charge transfer is associated with generation of EMF in the membrane, but independent of the effect of prestin on membrane mechanics. Based on our results, we propose synergistic effects of prestin and the membrane in the generation of NLC and electromotility. The information these results provide is important for understanding protein/membrane interactions, prestin properties and the origin of electromotility.
Optical characterization and in vivo assessment of charge-assembled capsules containing indocyanine green for laser-mediated diagnosis and therapy
(2008) Yaseen, Mohammad Abbas; Anvari, Bahman
We have developed a carrier system to encapsulate Indocyanine Green (ICG) to overcome its limitations for laser-mediated medical applications. Charge-assembled capsules with controllable size and adjustable coatings efficiently encapsulate ICG and effectively stabilize its optical properties. When encapsulated, ICG retains its pronounced light absorption and fluorescence properties in the near infrared spectral range. The capsules' in vivo circulation kinetics and biodistribution can be adjusted by coating the surface with various polymers or nanoparticles. The stability enhancement of ICG's optical properties by encapsulation within our capsule system was investigated using absorbance spectroscopy. The ability of capsules containing ICG to induce photothermolysis was demonstrated in an ex vivo tumorous tissue model. Biodistribution of capsules with different coating material and of different sizes was evaluated in a healthy mouse model. And the circulation kinetics and imaging quality of capsules with different coatings was assessed in mice using in vivo fluorescence imaging. Capsules containing ICG show promise for a variety of laser-mediated medical applications including phothothermal treatment of tumors and vascular lesions as well as fluorescence-based pulmonary imaging.
Optimization of cryogen spray cooling: Increasing epidermal protection during cutaneous laser procedures
(2004) Pikkula, Brian Matthew; Anvari, Bahman
Cryogen spray cooling (CSC) is an effective method to minimize epidermal damage during laser treatment of various cutaneous anomalies such as port wine stains (PWS). We examined the mechanism of heat removal by CSC from skin, and heat removal was compared for CSC and contact cooling, another precooling method for cutaneous laser therapy. Next, we examined the heat removal ability of various cryogen delivery devices using a skin phantom and an inverse heat conduction algorithm. Third, the droplet characteristics were evaluated and correlated with heat removal in the skin phantom. An ex vivo study was used to confirm the results of the phantom work. Lastly, a comparative study of the droplet characteristics used to increase heat removal was performed on in vivo human skin to verify results from previous studies. Results demonstrated that heat removal is limited in skin by its low thermal diffusivity and heat removal by evaporative cooling was negligible on skin using CSC. Next, it was determined that different cryogen delivery devices remove various amounts of heat from the skin phantom. The cause of the differing rates of heat removal was determined to be by three main factors: the initial temperature differential between the impinging droplets and substrate, velocity of the droplets, and droplet diameter. The Weber number, which is proportional to the velocity2·diameter, showed a correlation to heat removal in the skin phantom. Using an equal temperature differential, it was confirmed that an increased Weber number provided greater epidermal protection in ex vivo human skin in response to laser irradiation. Lastly, an in vivo study of human skin also demonstrated that an increased Weber number spray was better able to reduce nonspecific thermal damage, especially with darker skin types, in response to 595 nm pulsed laser irradiation. Gross purpura after irradiation was correlated to vascular damage histologically and to apoptosis, giving the clinician a visual indication of the vascular damage occurring. This study confirmed that although the low thermal diffusivity of skin is a limiting factor in heat removal, altering the droplet characteristics can increase heat removal and provide increased epidermal protection.
Quantification of staphylococcal adhesion using optical tweezers
(2004) Simpson, Kathryn Hicks; Anvari, Bahman
Biofilm formation, a common cause of medical device failure and tissue infection, often follows bacterial adhesion to proteins present in the tissue or adsorbed on the implant surface. Certain species of gram-positive bacteria have covalently anchored transmembrane molecules called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) which mediate adhesion. Developing a greater understanding of the interactions between MSCRAMMs and their ligands can lead to improved methods of combating bacterial adhesion. The goal of this research was to use optical tweezers to quantify and characterize the forces of staphylococcal detachment from surfaces coated with extracellular matrix (ECM) molecules. An optically trapped bacterium was brought in contact with an ECM-coated polystyrene microsphere, and the force required to separate the cell and microsphere was determined. The forces required to detach S. epidemidis from fibronectin occurred in a series of clusters whose means were integer multiples of an 18-piconewton (pN) base value depending on the number of bonds formed. For S. aureus binding, this estimated single-bond force was 25 pN for fibronectin and 20 pN for fibrinogen, respectively. In S. aureus, we have found that varying degrees of mutation of the fibronectin-binding MSCRAMM may cause reduction or inhibition of binding depending on the degree of mutation. However, multiple mutations are required before any reduction in binding is observed, which confirms that multiple regions of the S. aureus fibronectin MSCRAMM may substitute for one another in the binding process. We have also tracked the force required to detach S. aureus from fibronectin using the signal generated by the trapped cell on a quadrant photodiode throughout the detachment process for a series of loading rates. The magnitude of the force increased in an approximately linear fashion until the point of bond rupture. The peak single-bond rupture forces ranged from 10 to 29 pN for loading rates spanning three orders of magnitude from 101 to 103 pN/s. Bond lifetime increased with loading rate, which suggests the presence of a catch-bonding mechanism. This work provides additional insight into the specific binding mechanisms of staphylococci and is a step toward developing improved methods of preventing or treating infections.
Selective vascular injury during cutaneous laser therapy
(2003) Tunnell, James William; Anvari, Bahman
Pulsed laser irradiation in conjunction with cryogen spray cooling (CSC) can induce selective vascular injury to remove cutaneous hypervascular malformations such as port wine stains (PWS), hemangiomas, and facial veins. In this group of studies, we characterized the cryogen heat removal process and determined the effects of pulsed laser irradiation in conjunction with cryogen spray cooling of human skin. First, we employed an inverse heat conduction algorithm to measure the thermal boundary condition due to CSC in in vitro skin phantoms. Second, we developed a mathematical model of laser irradiation in conjunction with CSC in human skin. We determined tissue damage and temperature profiles due to varying combinations of laser pulse duration, radiant exposure, and CSC application times. Finally, we used ex vivo and in vivo human skin to determine the effects of high radiant exposures and CSC on epidermal and vascular injury. CSC induces a dynamic cooling effect, removing heat from the skin both during and following the spurt application time. Residual cryogen, deposited on the skin surface during the cryogen spurt, remains on the skin surface several times as long as the as cryogen spurt itself. The heat removal rate during the cryogen spurt is greatest; however, the total energy removed following the cryogen spurt is also substantial (approximately half as much as during the spurt application time). CSC was effective in protecting the human skin epidermis in light to moderately pigmented skin. Mathematical modeling, ex vivo, and in vivo studies showed that the epidermal damage threshold could be increased by a factor of approximately two. Increased radiant exposures increased the risk of non-selective vascular injury observed in histology as injury to the epidermis and perivascular collagen; however, proper choice of cryogen cooling durations resulted in the elimination of epidermal injury as well as perivascular tissue injury. In addition, higher radiant exposures induced vascular injury deeper within the dermis. Although cooling alone was not sufficient in protecting the epidermis in dark skin patients, the mathematical model suggests that selecting the proper pulse duration in conjunction with CSC may eliminate epidermal injury in darker skin patients.
Studies of platelet glycoprotein Ib-IX-V adhesion tovon Willebrand factor using optical tweezers
(2004) Arya, Maneesh; Anvari, Bahman
The first stage in hemostasis and thrombosis is the binding of the platelet membrane receptor, glycoprotein (GP) Ib-IX, to its ligand, von Willebrand factor (VWF), in the subendothelium. Using an optical tweezers system, we have measured the binding strength of: (1) VWF variants (unusually large VWF (ULVWF), plasma VWF, and isolated VWF A1 domain) to GP Ib-IX receptors; (2) GP Ib-IX variants (receptors with human-canine chimeric sequences in the leucine-rich domain, and truncated receptors) to plasma VWF; (3) plasma VWF to gain-of-function (GOF) and loss-of-function (LOF) mutated GP Ib-IX receptors; (4) VWF Al domain to clustered GP Ib-IX receptors; and (5) ULVWF multimers to GP Ib-IX in the presence of the VWF cleaving protease (ADAMTS-13) and other modulators. In addition, we dynamically measured unbinding force profiles between A1 and GP Ib-IX at loading rates ranging from 200--20,000 pN/s and examined the relationship between bond rupture force and loading rate. In the absence of shear stress, ULVWF multimers formed spontaneous high-strength bonds with GP Ib-IX, while plasma VWF required exogenous modulators. The strength of individual bonds formed with GP Ib-IX was similar for both ULVWF and the isolated A1-domain and greater than those of plasma VWF induced by an exogenous modulator. We observed ULVWF/GP Ib-IX binding was completely abolished with the addition of either ADAMTS-13 or plasma cryosupernatant. We found that the putative single bond strengths between A1 and GP Ibalpha GOF mutants were significantly greater than the A1/wild-type GP Ib-IX bond at all loading rates examined. In addition, the GP Ibalpha LOF mutants exhibited significantly lower putative single bond strengths with A1 than the wild-type receptors. The experiments involving the adhesion of either chimeric or truncated GP Ib-IX receptors to VWF illustrated the importance of various regions of the glycoprotein in properly binding VWF. The minimal detachment force between GP Ib-IX and A1 or plasma VWF doubled after an exogenous clustering agent was added, thereby demonstrating the importance of avidity, as opposed to affinity, modulation. Finally, we observed a linear relationship between the rupture force and the logarithm of the loading rate, consistent with the Bell Model.

Browsing by Author "Anvari, Bahman"

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