Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion

A method for cell–cell and cell–liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell–cell or cell–liposome pair was achieved by the application of a highly focused electric field through a pair of 5-μm o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell–liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (γ-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.

Electric field-induced reorganization of two-component supported bilayer membranes

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory’s approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi–Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.

Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.

Electric Signaling and Pin2 Gene Expression on Different Abiotic Stimuli Depend on a Distinct Threshold Level of Endogenous Abscisic Acid in Several Abscisic Acid-Deficient Tomato Mutants1

Experiments were performed on three abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) mutants, notabilis, flacca, and sitiens, to investigate the role of ABA and jasmonic acid (JA) in the generation of electrical signals and Pin2 (proteinase inhibitor II) gene expression. We selected these mutants because they contain different levels of endogenous ABA. ABA levels in the mutant sitiens were reduced to 8% of the wild type, in notabilis they were reduced to 47%, and in flacca they were reduced to 21%. In wild-type and notabilis tomato plants the induction of Pin2 gene expression could be elicited by heat treatment, current application, or mechanical wounding. In flacca and sitiens only heat stimulation induced Pin2 gene expression. JA levels in flacca and sitiens plants also accumulated strongly upon heat stimulation but not upon mechanical wounding or current application. Characteristic electrical signals evolved in the wild type and in the notabilis and flacca mutants consisting of a fast action potential and a slow variation potential. However, in sitiens only heat evoked electrical signals; mechanical wounding and current application did not change the membrane potential. In addition, exogenous application of ABA to wild-type tomato plants induced transient changes in membrane potentials, indicating the involvement of ABA in the generation of electrical signals. Our data strongly suggest the presence of a minimum threshold value of ABA within the plant that is essential for the early events in electrical signaling and mediation of Pin2 gene expression upon wounding. In contrast, heat-induced Pin2 gene expression and membrane potential changes were not dependent on the ABA level but, rather, on the accumulation of JA.

Molecular dynamics of a grid-mounted molecular dipolar rotor in a rotating electric field

Classical molecular dynamics is applied to the rotation of a dipolar molecular rotor mounted on a square grid and driven by rotating electric field E(ν) at T ≃ 150 K. The rotor is a complex of Re with two substituted o-phenanthrolines, one positively and one negatively charged, attached to an axial position of Rh\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{4+}}}\end{equation*}\end{document} in a [2]staffanedicarboxylate grid through 2-(3-cyanobicyclo[1.1.1]pent-1-yl)malonic dialdehyde. Four regimes are characterized by a, the average lag per turn: (i) synchronous (a < 1/e) at E(ν) = |E(ν)| > Ec(ν) [Ec(ν) is the critical field strength], (ii) asynchronous (1/e < a < 1) at Ec(ν) > E(ν) > Ebo(ν) > kT/μ, [Ebo(ν) is the break-off field strength], (iii) random driven (a ≃ 1) at Ebo(ν) > E(ν) > kT/μ, and (iv) random thermal (a ≃ 1) at kT/μ > E(ν). A fifth regime, (v) strongly hindered, W > kT, Eμ, (W is the rotational barrier), has not been examined. We find Ebo(ν)/kVcm−1 ≃ (kT/μ)/kVcm−1 + 0.13(ν/GHz)1.9 and Ec(ν)/kVcm−1 ≃ (2.3kT/μ)/kVcm−1 + 0.87(ν/GHz)1.6. For ν > 40 GHz, the rotor behaves as a macroscopic body with a friction constant proportional to frequency, η/eVps ≃ 1.14 ν/THz, and for ν < 20 GHz, it exhibits a uniquely molecular behavior.

Electric-field-induced Schiff-base deprotonation in D85N mutant bacteriorhodopsin.

The application of an external electric field to dry films of Asp-85-->Asn mutant bacteriorhodopsin causes deprotonation of the Schiff base, resulting in a shift of the optical absorption maximum from 600 nm to 400 nm. This is in marked contrast to the case of wild-type bacteriorhodopsin films, in which electric fields produce a red-shifted product whose optical properties are similar to those of the acid-blue form of the protein. This difference is due to the much weaker binding of the Schiff-base proton in the mutant protein, as indicated by its low pK of approximately 9, as compared with the value pK approximately 13 in the wild type. Other bacteriorhodopsins with lowered Schiff-base pK values should also exhibit a field-induced shift in the protonation equilibrium of the Schiff base. We propose mechanisms to account for these observations.

Rectification and signal averaging of weak electric fields by biological cells.

Oscillating electric fields can be rectified by proteins in cell membranes to give rise to a dc transport of a substance across the membrane or a net conversion of a substrate to a product. This provides a basis for signal averaging and may be important for understanding the effects of weak extremely low frequency (ELF) electric fields on cellular systems. We consider the limits imposed by thermal and "excess" biological noise on the magnitude and exposure duration of such electric field-induced membrane activity. Under certain circumstances, the excess noise leads to an increase in the signal-to-noise ratio in a manner similar to processes labeled "stochastic resonance." Numerical results indicate that it is difficult to reconcile biological effects with low field strengths.

Effects of the environment on the electric conductivity of double-stranded DNA molecules

We present a theoretical analysis of the effects of the environment on charge transport in double-stranded synthetic poly(G)-poly(C) DNA molecules attached to two ideal leads. Coupling of the DNA to the environment results in two effects: (i) localization of carrier functions due to static disorder and (ii) phonon-induced scattering of the carriers between the localized states, resulting in hopping conductivity. A nonlinear Pauli master equation for populations of localized states is used to describe the hopping transport and calculate the electric current as a function of the applied bias. We demonstrate that, although the electronic gap in the density of states shrinks as the disorder increases, the voltage gap in the I-V characteristics becomes wider. A simple physical explanation of this effect is provided.

A Community Analysis of the Implementation for Household Curbside Recycling Programs: A Diversionary Method to Control Municipal Solid Waste

The Municipality of Anchorage (MOA) is required to better manage, operate and control municipal solid waste (MSW) after the Anchorage Assembly instituted a Zero Waste Policy. Two household curbside recycling programs (CRPs), pay-as-you-throw (PAYT) and single-stream, were compared and evaluated to determine an optimal municipal solid waste diversion method for households within the MOA. The analyses find: (1) a CRP must be designed from comprehensive analysis, models and data correlation that combine demographic and psychographic variables; and (2) CRPs can be easily adjusted towards community-specific goals using technology, such as Geographic Information System (GIS) and Radio Frequency Identification (RFID). Combining resources of policy-makers, businesses, and other viable actors are necessary components to produce a sustainable, economically viable curbside recycling program.

Comprehensive Greenhouse Gas Management for the Electric Generating Industry

Climate change is becoming more visible in the political arena. Electric generating companies will likely be impacted by future regulation of climate change related emissions. Even though few climate related programs are mandatory, electric generating companies should begin to implement greenhouse gas management systems. This report includes a review of issues facing the electric generating industry, an examination of current emission management programs, and recommendations for an effective greenhouse gas management framework. An effective greenhouse gas management program allows a company to continually improve their impact on climate change by reducing emissions using the plan, do, check, act process. To ease the reporting burden, companies should apply de minimis exemptions to sources that produce less than 5% of emissions.

Opportunities and Challenges for the Widespread Implementation of Battery Powered Electric Transit Buses

The need for more sustainable public transportation choices drives innovation and provides opportunity for improvement in options. Transit buses provide many advantages for efficient transportation and electric drive vehicles are anticipated to play an increasing role in future transportation systems. A lifecycle cost analysis of battery electric transit buses indicates rate structures and demand charges do not currently have a large impact on lifecycle cost for small fleets of battery electric buses. As fleets grow, policies and rate structures will need to adjust to avoid becoming a barrier to adoption. Battery electric transit buses are now being developed which promise to address the primary issues of high life cycle cost, low reliability, range, and flexibility.

Development of a Household Pharmaceutical Waste Collection Program for Atlanta, Georgia: An Analysis of Risk Factors and Public Awareness with a Comprehensive Collection Plan

Decades of mixed messages from three federal agencies left many Americans unaware of the hazards associated with the indiscriminate disposal of unwanted or expired medicines. For this Capstone project, a systematic review of state and federal regulations was undertaken to determine how these laws obstruct household pharmaceutical waste collection. In addition, a survey of 654 Atlanta residents was conducted to evaluate unwanted medicine disposal habits, awareness of pharmaceutical compounds being detected in drinking water, surface, and ground waters, and willingness to participate in a household pharmaceutical waste collection program. Survey responses were tabulated to provide overall results and by age group, gender, and race. A household pharmaceutical waste collection plan was developed for the city and included as an appendix.

Invisible work, unseen hazards: the health of women immigrant household service workers in Spain

BACKGROUND: Household service work has been largely absent from occupational health studies. We examine the occupational hazards and health effects identified by immigrant women household service workers. METHODS: Exploratory, descriptive study of 46 documented and undocumented immigrant women in household services in Spain, using a phenomenological approach. Data were collected between September 2006 and May 2007 through focus groups and semi-structured individual interviews. Data were separated for analysis by documentation status and sorted using a mixed-generation process. In a second phase of analysis, data on psychosocial hazards were organized using the Copenhagen Psychosocial Questionnaire as a guide. RESULTS: Informants reported a number of environmental, ergonomic and psychosocial hazards and corresponding health effects. Psychosocial hazards were especially strongly present in data. Data on reported hazards were similar by documentation status and varied by several emerging categories: whether participants were primarily cleaners or carers and whether they lived in or outside of the homes of their employers. Documentation status was relevant in terms of empowerment and bargaining, but did not appear to influence work tasks or exposure to hazards directly. CONCLUSIONS: Female immigrant household service workers are exposed to a variety of health hazards that could be acted upon by improved legislation, enforcement, and preventive workplace measures, which are discussed.