David Wu

Reversing blood flows act through klf2a to ensure normal valvulogenesis in the developing heart.

The directionality of local blood flow in the zebrafish embryonic heart is essential for proper heart valve formation.

The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear

In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized ‘ear stones’ (or otoliths). Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.

A Single-Molecule Hershey-Chase Experiment

Ever since Hershey and Chase used phages to establish DNA as the carrier of genetic information in 1952, the precise mechanisms of phage DNA translocation have been a mystery. Although bulk measurements have set a timescale for in vivo DNA translocation during bacteriophage infection, measurements of DNA ejection by single bacteriophages have only been made in vitro. Here, we present direct visualization of single bacteriophages infecting individual Escherichia coli cells. For bacteriophage λ, we establish a mean ejection time of roughly 5 min with significant cell-to-cell variability, including pausing events. In contrast, corresponding in vitro single-molecule ejections are more uniform and finish within 10 s. Our data reveal that when plotted against the amount of DNA ejected, the velocity of ejection for two different genome lengths collapses onto a single curve. This suggests that in vivo ejections are controlled by the amount of DNA ejected. In contrast, in vitro DNA ejections are governed by the amount of DNA left inside the capsid. This analysis provides evidence against a purely intrastrand repulsion-based mechanism and suggests that cell-internal processes dominate. This provides a picture of the early stages of phage infection and sheds light on the problem of polymer translocation.

Mechanistic Basis of Otolith Formation during Teleost Inner Ear Development

Otoliths, which are connected to stereociliary bundles in the inner ear, serve as inertial sensors for balance. In teleostei, otolith development is critically dependent on flow forces generated by beating cilia; however, the mechanism by which flow controls otolith formation remains unclear. Here, we have developed a noninvasive flow probe using optical tweezers and a viscous flow model in order to demonstrate how the observed hydrodynamics influence otolith assembly. We show that rotational flow stirs and suppresses precursor agglomeration in the core of the cilia-driven vortex. The velocity field correlates with the shape of the otolith and we provide evidence that hydrodynamics is actively involved in controlling otolith morphogenesis. An implication of this hydrodynamic effect is that otolith self-assembly is mediated by the balance between Brownian motion and cilia-driven flow. More generally, this flow feature highlights an alternative biological strategy for controlling particle localization in solution.

Ion-Dependent Dynamics of DNA Ejections for Bacteriophage λ

We studied the control parameters that govern the dynamics of in vitro DNA ejection in bacteriophage lambda. Previous work demonstrated that bacteriophage DNA is highly pressurized, and this pressure has been hypothesized to help drive DNA ejection. Ions influence this process by screening charges on DNA; however, a systematic variation of salt concentrations to explore these effects has not been undertaken. To study the nature of the forces driving DNA ejection, we performed in vitro measurements of DNA ejection in bulk and at the single-phage level. We present measurements on the dynamics of ejection and on the self-repulsion force driving ejection. We examine the role of ion concentration and identity in both measurements, and show that the charge of counterions is an important control parameter. These measurements show that the mobility of ejecting DNA is independent of ionic concentrations for a given amount of DNA in the capsid. We also present evidence that phage DNA forms loops during ejection, and confirm that this effect occurs using optical tweezers.

Adaptive steering controller using a Kalman estimator for wheel-type agricultural tractors

This paper presents an adaptive steering controller for achieving accurate and prompt steering control with noisy steering command signals and drifting valve characteristics on an automated agricultural tractor with an electrohydraulic steering system. It is difficult to accomplish performance objectives with conventional PID controllers because of the effects of disturbances and unknown factors. The adaptive controller, consisted of an adaptive gain tuner and an adaptive nonlinearity compensator, was to overcome these performance obstacles. Test results indicated that this controller provided an effective means for achieving satisfactory steering control for automated tractor traveling on changing and unpredictable farm field courses.

Model recognition and validation for an off-road vehicle electrohydraulic steering controller

Automated steering control is essential for an autonomous off-road vehicle. Many off-road vehicles use an electrohydraulic (E/H) actuator to implement the steering control. This paper reports the design and validation of an electrohydraulic steering controller through a combination of system identification, model simulation, and field tests. A kinematic model of the steering linkage geometry provided the gain between the hydraulic actuator and the front wheels. The system model was used to close the steering control loop based on the feedback signal from the hydraulic steering actuator rather than from the front wheels. Test results were used to identify the non-linear and dynamic characteristics of the original electrohydraulic steering system. The system identification model was used to develop a preliminary controller, which was simulated under Matlab before beginning full-scale vehicle testing. The simulation and vehicle test results indicated that the steering controller developed was capable of efficiently handling the non-linearity and dynamic asymmetry of the electrohydraulic steering system.