The CaMKII␣ mRNA extends into distal hippocampal dendrites, and the 3Ј untranslated region (3ЈUTR) is sufficient to mediate this localization. We labeled the 3ЈUTR of the CaMKII␣ mRNA in hippocampal cultures by using a green fluorescent protein (GFP)/ MS2 bacteriophage tagging system. The CaMKII␣ 3ЈUTR formed discrete granules throughout the dendrites of transfected cells. The identity of the fluorescent granules was verified by in situ hybridization. Over 30 min time periods these granules redistributed without a net increase in granule number; with depolarization there is a tendency toward increased numbers of granules in the dendrites. These observations suggest that finer time resolution of granule motility might reveal changes in the motility characteristics of granules after depolarization. So that motile granules could be tracked, shorter periods of observation were required. The movements of motile granules can be categorized as oscillatory, unidirectional anterograde, or unidirectional retrograde. Colocalization of CaMKII␣ 3ЈUTR granules and synapses suggested that oscillatory movements allowed the granules to sample several local synapses. Neuronal depolarization increased the number of granules in the anterograde motile pool. Based on the time frame over which the granule number increased, the translocation of granules may serve to prepare the dendrite for mounting an adequate local translation response to future stimuli. Although the resident pool of granules can respond to signals that induce local translation, the number of granules in a dendrite might reflect its activation history.

In the magnesium ion-dependent folding of the Tetrahymena ribozyme, a kinetic intermediate accumulates in which the P4-P6 domain is formed, but the P3-P7 domain is not. The kinetic barriers to P3-P7 formation were investigated with the use of in vitro selection to identify mutant RNA molecules in which the folding rate of the P3-P7 domain was increased. The critical mutations disrupt native tertiary interactions within the P4-P6 domain and increase the rate of P3-P7 formation by destabilizing a kinetically trapped intermediate. Hence, kinetic traps stabilized by native interactions, and not simply by mispaired nonnative structures, can present a substantial barrier to RNA folding.

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In this article we introduce a novel technology that utilizes specialized water dissolvable thin films for valving in centrifugal microfluidic systems. In previous work (William Meathrel and Cathy Moritz, IVD Technologies, 2007), dissolvable films (DFs) have been assembled in laminar flow devices to form efficient sacrificial valves where DFs simply open by direct contact with liquid. Here, we build on the original DF valving scheme to leverage sophisticated, merely rotationally actuated vapour barriers and flow control for enabling comprehensive assay integration with low-complexity instrumentation on "lab-on-a-disc" platforms. The advanced sacrificial valving function is achieved by creating an inverted gas-liquid stack upstream of the DF during priming of the system. At low rotational speeds, a pocket of trapped air prevents a surface-tension stabilized liquid plug from wetting the DF membrane. However, high-speed rotation disrupts the metastable gas/liquid interface to wet the DF and thus opens the valve. By judicious choice of the radial position and geometry of the valve, the burst frequency can be tuned over a wide range of rotational speeds nearly 10 times greater than those attained by common capillary burst valves based on hydrophobic constrictions. The broad range of reproducible burst frequencies of the DF valves bears the potential for full integration and automation of comprehensive, multi-step biochemical assay protocols. In this report we demonstrate DF valving, discuss the biocompatibility of using the films, and show a potential sequential valving system including the on-demand release of on-board stored liquid reagents, fast centrifugal sedimentation and vigorous mixing; thus providing a viable basis for use in lab-on-a-disc platforms for point-of-care diagnostics and other life science applications.