Use of CdS quantum dot-functionalized cellulose nanocrystal films for anti-counterfeiting applications.

Structural colors and photoluminescence have been widely used for anti-counterfeiting and security applications. We report for the first time the use of CdS quantum dot (QD)-functionalized cellulose nanocrystals (CNCs) as building blocks to fabricate nanothin films via layer-by-layer (LBL) self-assembly for anti-counterfeiting applications. Both negatively- and positively-charged CNC/QD nanohybrids with a high colloidal stability and a narrow particle size distribution were prepared. The controllable LBL coating process was characterized by scanning electron microscopy and ellipsometry. The rigid structure of CNCs leads to nanoporous structured films on poly(ethylene terephthalate) (PET) substrates with high transmittance (above 70%) over the entire range of visible light and also resulted in increased hydrophilicity (contact angles of ∼40 degrees). Nanothin films on PET substrates showed good flexibility and enhanced stability in both water and ethanol. The modified PET films with structural colors from thin-film interference and photoluminescence from QDs can be used in anti-counterfeiting applications.

Synthesis and characterization of pH-responsive and fluorescent poly (amidoamine) dendrimer-grafted cellulose nanocrystals.

Poly (amidoamine) (PAMAM) dendrimers have found promising applications in biomedicine and in the encapsulation of inorganic nanoparticles. G6 PAMAM dendrimer-grafted cellulose nanocrystals (CNC-PAMAM) were prepared via a simple carbodiimide-mediated amidation process and they displayed pH-responsive and fluorescent characteristics as confirmed by zeta potential, transmittance, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Stable aqueous dispersions of CNC-PAMAM were obtained at pH⩽4 and pH⩾10, driven by electrostatic repulsion from positive charge and negative charge respectively. However, large aggregates were formed at pH values from 5 to 9 due to electrostatic attraction. In addition, strong blue fluorescent emission was observed, and the fluorescent behaviour of CNC-PAMAM was influenced by the formation of aggregates. The pH-responsive and fluorescent properties of CNC-PAMAM may be suitable for their applications in pH-responsive nanodevices, fluorescent-based pH sensors, optical markers, and nanoreactors for the encapsulation of inorganic nanoparticles.

Controlled delivery of membrane proteins to artificial lipid bilayers by nystatin-ergosterol modulated vesicle fusion.

The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render protein-carrying vesicles fusogenic. After fusion with an ergosterol-free artificial bilayer, the nystatin-ergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid species was reduced. Under these conditions, the -amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems.

Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication.

Practical components for three-dimensional molecular nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nanometers on a side, that can self-assemble in seconds with near-quantitative yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an atomic force microscope, we have measured the axial compressibility of DNA and observed the buckling of the double helix under high loads.

Theories of rotary motors.

The bacterial flagellar motor and the ATP-hydrolysing F1 portion of the F1Fo-ATPase are known to be rotary motors, and it seems highly probable that the H+-translocating Fo portion rotates too. The energy source in the case of Fo and the flagellar motor is the flow of ions, either H+ (protons) or Na+, down an electrochemical gradient across a membrane. The fact that ions flow in a particular direction through a well-defined structure in these motors invites the possibility of a type of mechanism based on geometric constraints between the rotor position and the paths of ions flowing through the motor. The two best-studied examples of such a mechanism are the 'turnstile' model of Khan and Berg and the 'proton turbine' model of Läuger or Berry. Models such as these are typically represented by a small number of kinetic states and certain allowed transitions between them. This allows the calculation of predictions of motor behaviour and establishes a dialogue between models and experimental results. In the near future structural data and observations of single-molecule events should help to determine the nature of the mechanism of rotary motors, while motor models must be developed that can adequately explain the measured relationships between torque and speed in the flagellar motor.

Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio.

Rotation of the bacterial flagellar motor is driven by an ensemble of torque-generating units containing the proteins MotA and MotB. Here, by inducing expression of MotA in motA- cells under conditions of low viscous load, we show that the limiting speed of the motor is independent of the number of units: at vanishing load, one unit turns the motor as rapidly as many. This result indicates that each unit may remain attached to the rotor for most of its mechanochemical cycle, that is, that it has a high duty ratio. Thus, torque generators behave more like kinesin, the protein that moves vesicles along microtubules, than myosin, the protein that powers muscle. However, their translation rates, stepping frequencies and power outputs are much higher, being greater than 30 microm s(-1), 12 kHz and 1.5 x 10(5) pN nm s(-1), respectively.

Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity.

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.

The bacterial flagella motor.

The bacterial flagellum is probably the most complex organelle found in bacteria. Although the ribosome may be made of slightly more subunits, the bacterial flagellum is a more organized and complex structure. The limited number of flagella must be targeted to the correct place on the cell membrane and a structure with cytoplasmic, cytoplasmic membrane, outer membrane and extracellular components must be assembled. The process of controlled transcription and assembly is still not fully understood. Once assembled, the motor complex in the cytoplasmic membrane rotates, driven by the transmembrane ion gradient, at speeds that can reach many 100 Hz, driving the bacterial cell at several body lengths a second. This coupling of an electrochemical gradient to mechanical rotational work is another fascinating feature of the bacterial motor. A significant percentage of a bacterium's energy may be used in synthesizing the complex structure of the flagellum and driving its rotation. Although patterns of swimming may be random in uniform environments, in the natural environment, where cells are confronted with gradients of metabolites and toxins, motility is used to move bacteria towards their optimum environment for growth and survival. A sensory system therefore controls the switching frequency of the rotating flagellum. This review deals primarily with the structure and operation of the bacterial flagellum. There has been a great deal of research in this area over the past 20 years and only some of this has been included. We apologize in advance if certain areas are covered rather thinly, but hope that interested readers will look at the excellent detailed reviews on those areas cited at those points.

Torque generated by the flagellar motor of Escherichia coli while driven backward.

The technique of electrorotation was used to apply torque to cells of the bacterium Escherichia coli tethered to glass coverslips by single flagella. Cells were made to rotate backward, that is, in the direction opposite to the rotation driven by the flagellar motor itself. The torque generated by the motor under these conditions was estimated using an analysis that explicitly considers the angular dependence of both the viscous drag coefficient of the cell and the torque produced by electrorotation. Motor torque varied approximately linearly with speed up to over 100 Hz in either direction, placing constraints on mechanisms for torque generation in which rates of proton transfer for backward rotation are limiting. These results, interpreted in the context of a simple three-state kinetic model, suggest that the rate-limiting step in the torque-generating cycle is a powerstroke in which motor rotation and dissipation of the energy available from proton transit occur synchronously.

Genetic enhancement in the twenty-first century: three problems in legal imagining.

By what method should we resolve the ethical and public policy issues surrounding genetic enhancement: a utilitarian calculus, appeal to Scripture, application of neo-Kantian principles? In this Article, the author claims that, despite the diversity of our basic methodological commitments, we can advance our shared understanding of the issues surrounding genetic enhancement and come to agree upon certain central features as essential to any adequate method for resolving those issues. The author attempts to make modest headway on both fronts by engaging in "legal imagining." Legal imagining consists of examining hypothetical problems from the lawyer's perspective, the perspective of a caring, professional friend. This Article examines and discusses three problems in legal imagining.

Absence of a barrier to backwards rotation of the bacterial flagellar motor demonstrated with optical tweezers.

A cell of the bacterium Escherichia coli was tethered covalently to a glass coverslip by a single flagellum, and its rotation was stopped by using optical tweezers. The tweezers acted directly on the cell body or indirectly, via a trapped polystyrene bead. The torque generated by the flagellar motor was determined by measuring the displacement of the laser beam on a quadrant photodiode. The coverslip was mounted on a computer-controlled piezo-electric stage that moved the tether point in a circle around the center of the trap so that the speed of rotation of the motor could be varied. The motor generated approximately 4500 pN nm of torque at all angles, regardless of whether it was stalled, allowed to rotate very slowly forwards, or driven very slowly backwards. This argues against models of motor function in which rotation is tightly coupled to proton transit and back-transport of protons is severely limited.

Torque generated by the bacterial flagellar motor close to stall.

In earlier work in which electrorotation was used to apply external torque to tethered cells of the bacterium Escherichia coli, it was found that the torque required to force flagellar motors backward was considerably larger than the torque required to stop them. That is, there appeared to be substantial barrier to backward rotation. Here, we show that in most, possibly all, cases this barrier is an artifact due to angular variation of the torque applied by electrorotation, of the motor torque, or both; the motor torque appears to be independent to speed or to vary linearly with speed up to speeds of tens of Hertz, in either direction. However, motors often break catastrophically when driven backward, so backward rotation is not equivalent to forward rotation. Also, cells can rotate backward while stalled, either in randomly timed jumps of 180 degrees or very slowly and smoothly. When cells rotate slowly and smoothly backward, the motor takes several seconds to recover after electrorotation is stopped, suggesting that some form of reversible damage has occurred. These findings do not affect the interpretation of electrorotation experiments in which motors are driven rapidly forward.

Mechanical limits of bacterial flagellar motors probed by electrorotation.

We used the technique of electrorotation to apply steadily increasing external torque to tethered cells of the bacterium Escherichia coli while continuously recording the speed of cell rotation. We found that the bacterial flagellar motor generates constant torque when rotating forward at low speeds and constant but considerably higher torque when rotating backward. At intermediate torques, the motor stalls. The torque-speed relationship is the same in both directional modes of switching motors. Motors forced backward usually break, either suddenly and irreversibly or progressively. Motors broken progressively rotate predominantly at integral multiples of a unitary speed during the course of both breaking and subsequent recovery, as expected if progressive breaking affects individual torque-generating units. Torque is reduced by the same factor at all speeds in partially broken motors, implying that the torque-speed relationship is a property of the individual torque-generating units.

Defective escape mutants of HIV.

The virological literature presents two broad types of defective virus mutants that can alter the outcome of viral infection. In some infections, defective interfering particles reduce the replication of wild-type virus and lead to an attenuated or persistent infection. In other cases, very specific and highly pathogenic defective mutants lead to virulent disease in the presence of a much less pathogenic but replication-competent helper virus. Here, we outline the theoretical possibility that defective mutants of HIV, which escape from some of the immune responses directed at the wild-type virus, can have a positive effect on total virus growth in HIV infections. The high error rate of HIV may generate many mutants that have some altered epitope (escape mutants), but at the cost of greatly reduced or completely impaired reproductive abilities. If these mutants retain some ability to impair immune cell function, then the production of such "defective escape" mutants may enhance overall virus reproduction. This will be illustrated by a mathematical model.

Torque and switching in the bacterial flagellar motor. An electrostatic model.

A model is presented for the rotary motor that drives bacterial flagella, using the electrochemical gradient of protons across the cytoplasmic membrane. The model unifies several concepts present in previous models. Torque is generated by proton-conducting particles around the perimeter of the rotor at the base of the flagellum. Protons in channels formed by these particles interact electrostatically with tilted lines of charges on the rotor, providing "loose coupling" between proton flux and rotation of the flagellum. Computer simulations of the model correctly predict the experimentally observed dynamic properties of the motor. Unlike previous models, the motor presented here may rotate either way for a given direction of the protonmotive force. The direction of rotation only depends on the level of occupancy of the proton channels. This suggests a novel and simple mechanism for the switching between clockwise and counterclockwise rotation that is the basis of bacterial chemotaxis.

Correlated ion flux through parallel pores: application to channel subconductance states.

Many ion channels that normally gate fully open or shut have recently been observed occasionally to display well-defined subconductance states with conductances much less than those of the fully open channel. One model of this behavior is a channel consisting of several parallel pores with a strong correlation between the flux in each pore such that, normally, they all conduct together but, under special circumstances, the pores may transfer to a state in which only some of them conduct. This paper introduces a general technique for modeling correlated pores, and explores in detail by computer simulation a particular model based upon electric interaction between the pores. Correlation is obtained when the transient electric field of ions passing through the pores acts upon a common set of ionizable residues of the channel protein, causing transient changes in their effective pK and hence in their charged state. The computed properties of such a correlated parallel pore channel with single occupation of each pore are derived and compared to those predicted for a single pore that can contain more than one ion at a time and also to those predicted for a model pore with fluctuating barriers. Experiments that could distinguish between the present and previous models are listed.